Apelin polypeptides

ABSTRACT

The invention provides modified apelin polypeptides having increased stability, circulating half-life, and/or potency relative to the native apelin-13 polypeptide. Compositions comprising the modified apelin polypeptides and methods of using the polypeptides for treating cardiac disorders, such as heart failure, are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/317,911, filed on Dec. 9, 2016, which is a national stageapplication under 35 U.S.C. § 371 of International Application No.PCT/US2015/035205, having an international filing date of 10 Jun. 2015,which claims the benefit of, and priority to, U.S. Provisional PatentApplication No. 62/010,322, filed Jun. 10, 2014, which specification ishereby incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The application relates to agonists of the APJ receptor that haveincreased stability, circulating half-life and/or potency relative tonative apelin.

BACKGROUND OF THE INVENTION

Apelin is the endogenous ligand for the APJ receptor (APLNR, angiotensinreceptor like-1). The APJ receptor is a member of the rhodopsin-like Gprotein-coupled receptor (GPCR) family. The apelin/APJ system has beenobserved in many tissues such as heart, kidney, pancreas, lung and thecentral nervous system, suggesting diverse roles of the system in thephysiology and pathology of mammals.

Apelin peptides are processed from a 77 residue pre-pro form intosmaller bioactive fragments, mainly a 36 residue form (Apelin 42-77—alsoreferred to as Apelin-36) and a smaller 13 residue polypeptide (Apelin65-77—also referred to as Apelin-13) (Hosoya et al., J. Biol. Chem.275:21061-21067, 2000). Apelin peptides were previously determined to beendogenous ligands for the orphan APJ receptor, a member of the seventransmembrane G-protein-coupled receptor superfamily (Tatemoto et al.,Biochem. Biophys. Res. Commun., 251:471-476, 1998). One of the shortermore active isoforms identified, pyroglutamated apelin-13([PE65]Apelin-13 (65-77)), has been reported to be the most potent andabundant form of apelin in cardiac tissue (Maguire et al., Hypertension,54:598-604, 2009). In vitro and preclinical models have suggested thatthe apelin/APJ system has a role in cardiovascular homeostasis as wellas metabolism (Barnes et al., Heart, 96:1011-1016, 2010). Circulatingapelin levels are transient and Apelin-13 has a brief plasma half-lifeof <5 minutes leading to short-lived cardiovascular effects.

In vitro, exogenous apelin increases contractility at subnanomolarconcentrations in atrial strips and whole rat hearts, and increasessarcomere shortening by up to 140% in isolated cardiomyocytes (Barnes etal., Heart, 96:1011-1016, 2010). Apelin also has a potent inotropiceffect in an ex-vivo isolated heart assay. In vivo, acute apelininfusion restores ejection fraction, increases cardiac output andreduces left ventricular end-diastolic pressure in rats with chronicheart failure (Berry et al., Circulation, 110:187-193, 2004). Exogenousapelin potently enhances myocardial contractility without inducing leftventricular hypertrophy concomitant with reduction in ventricularpreload and afterload (Barnes et al., Heart, 96:1011-1016, 2010).

There have been very limited structure activity relationship (SAR)studies reported for apelin peptides. The limited SAR studies havefocused on in vitro affinity and/or potency enhancement. There are noreports with SAR focused on increasing proteolytic stability andprolonging circulating half-lives while improving or maintaining potencyof APJ agonists.

Thus, there is a need in the art for novel apelin analogs that retainthe potency of native apelin polypeptides but have increased stability.

SUMMARY OF THE INVENTION

The present invention provides novel modified apelin polypeptides thathave APJ agonist activity. In some embodiments, the modified peptideshave increased stability relative to wild-type apelin. The modifiedpeptides of the invention can be used to treat heart failure or otherdisorders that respond to activation of the APJ receptor.

In certain embodiments, the modified apelin polypeptides haveadvantageous pharmaceutical characteristics, such as increasedstability, half-life, and/or potency. In some embodiments, the modifiedapelin polypeptides comprise at least one D-amino acid, a β-amino acid,an N-methyl amino acid, an α-methyl amino acid, a non-canonical aminoacid or the D- or β-form of the non-canonical amino acid. In oneembodiment, the modified apelin polypeptide comprises two or more, threeor more, four or more, five or more, six or more, seven or more, oreight or more non-canonical amino acid residues. In certain embodiments,the non-canonical amino acid residues replace canonical amino acidresidues within the apelin polypeptide sequence. The modified apelinpolypeptides may be cyclized to further enhance stability.

In one embodiment, the apelin polypeptide comprises the amino acidsequence: X₁ X₂ X₃X₄ X₅ X₆ X₇ X₈ X₉X₁₀GX₁₁X₁₂ X₁₃ X₁₄ (SEQ ID NO: 717),wherein: X₁ is R, E, [hArg], or absent; X₂ is [r], R, E, [hArg], orabsent; X₃ is Q, [q], or [BLeu]; X₄ is [hArg], [NMeArg], R, E, or [r];X₅ is P or [aMePro]; X₆ is R, E, [r], [hArg] or [NMeArg]; X₇ is L,[aMeLeu], [BLeu], [NMeLeu] or [Cha]; X₈ is S, [BhSer], or [NhSerG]; X₉is H or Y; X₁₀ is K or [NLysG]; X₁₁ is P, [Oic], [aMePro], or [Pip]; X₁₂is [Nle], [rNle], or [pI-Phe]; X₁₃ is P, [BhPro], [aMePro], or [Aib];and X₁₄ is F, [D-BhPhe], [4-Cl-F], [D-4ClF], or [D-Bip]. In someembodiments, X₇ is [NMeLeu], X₁₂ is [pI-Phe], and X₁₄ is [D-Bip]. Incertain embodiments, X₁ is [hArg], X₂ is [hArg], X₃ is Q, X₄ is [hArg],and X₅ is P. In other embodiments, X₁₃ is [BhPro], [aMePro], or [Aib]and X₁₄ is [D-BhPhe] or [4-Cl-F]. In certain embodiments, X₆ is [NMeArg]or [hArg] and X₇ is [aMeLeu] or [BLeu]. The modified apelin polypeptidemay be acetylated at its amino terminus. The apelin polypeptidepreferably has very little or no antagonist activity against the APJreceptor (i.e. the apelin polypeptide is a full APJ receptor agonist).

In some embodiments, the modified apelin polypeptides may be conjugated,optionally through a linker, to another moiety, such as a fatty acid orother lipid, a polymer (e.g. polyethylene glycol polymer), protein (e.g.an antibody or Fc domain), or another peptide sequence (e.g. a targetingdomain) that serves as a half-life extension moiety. These half-lifeextension moiety-apelin peptide conjugates may have one or more improvedproperties, such as stability, in vivo half-life, or potency, relativeto native apelin peptides. The moiety may be conjugated to the apelinpolypeptide through the N- or C-terminus, or any other site of thepolypeptide. In certain embodiments, the half-life extension moiety isconjugated to the N-terminus of the modified apelin polypeptide.

In some embodiments, the modified apelin polypeptides or conjugatesthereof have increased in vitro or in vivo stability relative to nativeApelin-13 (SEQ ID NO: 4) or pyr-Apelin-13 (SEQ ID NO: 6). In variousembodiments, the modified apelin polypeptide with increased stabilityhas an increased in vitro half-life. In other embodiments, the modifiedapelin polypeptide with increased stability has an increased in vivohalf-life. The increased in vivo half-life may be a result of decreasedproteolysis or increased metabolic stability and/or inclusion of ahalf-life extension moiety or other modifications of the apelinpolypeptides.

The present invention also includes pharmaceutical compositionscomprising the modified apelin polypeptides or conjugates thereofdescribed herein and a pharmaceutically acceptable carrier. Thepharmaceutical compositions can be administered to a subject in need oftreatment for a cardiovascular disorder, such as heart failure.

Thus, in another embodiment, the present invention provides methods oftreating a cardiovascular disorder, improving cardiac contractility, orincreasing ejection fraction in a subject in need thereof comprisingadministering to the subject a pharmaceutical composition comprising atherapeutically effective amount of any of the modified apelinpolypeptides or conjugates thereof described herein. In someembodiments, the cardiovascular disorder is heart failure. The heartfailure may be acute heart failure or chronic heart failure (e.g.,chronic systolic or chronic diastolic). In one embodiment, the heartfailure is heart failure with reduced ejection fraction. In anotherembodiment, the heart failure is heart failure with preserved ejectionfraction. In other embodiments, the cardiovascular condition ishypertension.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Structures of exemplary non-canonical amino acids.

FIG. 2 . Examples of PEG groups and linker groups that can be attachedto apelin polypeptides of the invention.

FIG. 3 . Strategies to stabilize peptide agonists against metabolismusing backbone modifications.

FIGS. 4A-4D show inotropic effects of apelin polypeptide SEQ ID NO: 16(N=8) as compared with vehicle (N=6) in isolated perfused rat hearts.FIG. 4A shows percentage change over vehicle in left ventricularsystolic pressure. FIG. 4B shows percentage change over vehicle indeveloped left ventricular pressure. FIG. 4C shows percentage changeover vehicle in maximum rate of pressure change in the left ventricle.FIG. 4D shows percentage change over vehicle in minimum rate of pressurechange in the left ventricle. 2 out of 8 rats did not respond, but dataincludes all 8 rats. One way ANOVA analysis: ****<0.0001.

FIGS. 5A-5D show inotropic effects of apelin polypeptide SEQ ID NO: 53(N=8) as compared with vehicle (N=7) in isolated perfused rat hearts.FIG. 5A shows percentage change over vehicle in developed leftventricular pressure. FIG. 5B shows percentage change over vehicle inmaximum rate of pressure change in the left ventricle. FIG. 5C showspercentage change over vehicle in left ventricular systolic pressure.FIG. 5D shows percentage change over vehicle in minimum rate of pressurechange in the left ventricle.

FIGS. 6A-6D show the pharmacodynamic effects of pyr-apelin13 (SEQ ID NO:6) in rats with heart failure. FIG. 6A shows percentage change frombaseline in ejection fraction (EF) at three different dosages ofpyr-apelin. FIG. 6B shows percentage change from baseline in maximumrate of pressure change in the left ventricle (dP/dtmax) at threedifferent dosages of pyr-apelin. FIG. 6C shows percentage change frombaseline in mean arterial pressure (MAP) at three different dosages ofpyr-apelin. FIG. 6D shows percentage change from baseline in heart rate(HR) at three different dosages of pyr-apelin.

FIGS. 7A-7D show the pharmacodynamic effects of apelin polypeptide SEQID NO: 109 in rats with heart failure. FIG. 7A shows percentage changefrom baseline in ejection fraction (EF) at two different dosages of theapelin polypeptide. FIG. 7B shows percentage change from baseline inmaximum rate of pressure change in the left ventricle (dP/dtmax) at twodifferent dosages of the apelin polypeptide. FIG. 7C shows percentagechange from baseline in heart rate (HR) at two different dosages of theapelin polypeptide. FIG. 7D shows percentage change from baseline inmean arterial pressure (MAP) at two different dosages of the apelinpolypeptide.

FIGS. 8A-8D show the pharmacodynamic effects of apelin polypeptide SEQID NO: 16 in rats with heart failure. FIG. 8A shows percentage changefrom baseline in ejection fraction (EF) at two different dosages of theapelin polypeptide. FIG. 8B shows percentage change from baseline inmaximum rate of pressure change in the left ventricle (dP/dtmax) at twodifferent dosages of the apelin polypeptide. FIG. 8C shows percentagechange from baseline in mean arterial pressure (MAP) at two differentdosages of the apelin polypeptide.

FIG. 8D shows percentage change from baseline in heart rate (HR) at twodifferent dosages of the apelin polypeptide.

FIG. 9 shows the in vivo pharmacokinetics of four different 20 kDaPEGylated apelin peptides (SEQ ID NOs: 103, 105, 107, and 108) in rat. Aplot of concentration of peptide in rat plasma versus time is shown.Data for a dose of 5 mg/kg for SEQ ID NO: 103 (diamonds) was simulatedbased on predictive scaling to highlight the increased exposureresulting from the improved metabolic stability for this polypeptide.

FIGS. 10A-10B show the in vivo pharmacokinetics for various lipidatedapelin peptides in rat. FIG. 10A shows the plasma concentrations overtime for different lipids conjugated to the same peptide sequence. FIG.10B shows the plasma concentrations over time for different lipidsconjugated to different peptide sequences.

DETAILED DESCRIPTION

The foregoing summary is not intended to define every aspect orembodiment of the invention, and additional aspects may be described inother sections. The entire document is intended to be related as aunified disclosure, and it should be understood that all combinations offeatures described herein may be contemplated, even if the combinationof features is not found together in the same sentence, or paragraph, orsection of this document.

In addition to the foregoing, as an additional aspect, all embodimentsnarrower in scope in any way than the variations defined by specificparagraphs herein can be included in this disclosure. For example,certain aspects are described as a genus, and it should be understoodthat every member of a genus can be, individually, an embodiment. Also,aspects described as a genus or selecting a member of a genus should beunderstood to embrace combinations of two or more members of the genus.It should also be understood that while various embodiments in thespecification are presented using “comprising” language, under variouscircumstances, a related embodiment may also be described using“consisting of” or “consisting essentially of” language.

It will be understood that the descriptions herein are exemplary andexplanatory only and are not restrictive of the invention as claimed. Inthis application, the use of the singular includes the plural unlessspecifically stated otherwise. In this application, the use of “or”means “and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included,”is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one subunit unless specificallystated otherwise. Also, the use of the term “portion” can include partof a moiety or the entire moiety.

Unless otherwise defined herein, scientific and technical terms used inconnection with the application shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Thus, as usedin this specification and the appended claims, the singular forms “a”,“an”, and “the” include plural referents unless the context clearlyindicates otherwise. For example, reference to “a protein” includes aplurality of proteins; reference to “a cell” includes populations of aplurality of cells.

It should also be understood that when describing a range of values, thecharacteristic being described could be an individual value found withinthe range. For example, “a pH from about pH 4 to about pH 6,” could be,but is not limited to, pH 4, 4.2, 4.6, 5.1, 5.5, etc. and any value inbetween such values. Additionally, “a pH from about pH 4 to about pH 6,”should not be construed to mean that the pH in question varies 2 pHunits from pH 4 to pH 6, but rather a value may be picked from within atwo pH range for the pH of the solution.

In some embodiments, when the term “about” is used, it means the recitednumber plus or minus 1%, 5%, 10%, 15% or more of that recited number.The actual variation intended is determinable from the context.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

The present invention provides modified apelin polypeptides that act asagonists of the APJ receptor. As used herein, an “APJ receptor agonist”refers to a molecule that is able to activate the APJ receptor, whichcan then affect or stimulate biochemical, cellular or physiologicprocesses. The APJ receptor agonist may bind to the APJ receptor. Theterm also can refer to a polypeptide having biological activity at leastcomparable to a naturally-occurring apelin peptide; however, it can alsorefer to a polypeptide having biological activity less than anaturally-occurring apelin peptide. The term further includes moleculesthat potentiate the effects of the naturally-occurring apelin peptides.The APJ receptor agonist can be an APJ ligand, which refers to amolecule that binds to the APJ receptor or forms a complex with the APJreceptor. The ligand may be, but is not necessarily, a signal triggeringmolecule.

In some embodiments, the modified apelin polypeptides of the inventionare more stable as compared with native apelin polypeptides. Forinstance, in one embodiment, the modified apelin polypeptides of theinvention have increased stability (e.g. proteolytic stability) relativeto apelin-13 (SEQ ID NO: 4) or pyroglutamated apelin-13 (SEQ ID NO: 6).In these and other embodiments, the modified apelin polypeptides havecomparable or enhanced APJ receptor agonist activity as compared withnative apelin polypeptides. In certain embodiments, the modified apelinpolypeptides of the invention have an agonistic activity on the heart byincreasing, for example, various aspects of cardiac contractility, suchas dp/dt, ejection fraction, or heart rate. The term “native apelinpeptides” or “endogenous apelin peptides” includes full-length apelinpreprotein having the sequence MNLRLCVQAL LLLWLSLTAV CGGSLMPLPDGNGLEDGNVR HLVQPRGSRN GPGPWQGGRR KFRRQRPRLS HKGPMPF (SEQ ID NO: 2);apelin (42-77), also referred to as Apelin-36, having the sequenceLVQPRGSRNG PGPWQGGRRK FRRQRPRLSH KGPMPF (SEQ ID NO: 3); apelin (65-77),also referred to as wild-type Apelin-13, having the sequenceQRPRLSHKGPMPF (SEQ ID NO: 4); apelin (61-77), also known as Apelin-17,having the sequence KFRRQRPRLS HKGPMPF (SEQ ID NO:5), and otherfragments of full-length apelin. The apelin fragments are often definedas a particular range of amino acid sequences within the full-lengthapelin preprotein (SEQ ID NO: 2). For example, for Apelin-17, theN-terminal K is at position 61 and the C-terminal F is at position 77relative to the full-length apelin protein (SEQ ID NO: 2). In someembodiments, the modified apelin peptides of the invention may bedefined as having particular amino acid substitutions at positionscorresponding to amino acids 61 to 77 in the full-length apelin sequence(SEQ ID NO: 2). By way of example, the notation Oic74 refers to an Oicresidue in the modified polypeptide at a position corresponding to aminoacid 74 in SEQ ID NO: 2.

In one embodiment of the invention, the modified apelin polypeptidecomprises the sequence of the following formula:Nx¹x²x³x⁴x⁵x⁶x⁷x⁸x⁹x¹⁰x¹¹x¹²x¹³x¹⁴x¹⁵x¹⁶x¹⁷  (SEQ ID NO: 1),wherein:

N is an extension, conjugation linker or any moiety (e.g. an acetylgroup, γ-glu or a lipid) that can improve stability relative towild-type apelin-13 (SEQ ID NO: 4);

x¹ is absent, is a basic or polar amino acid residue, or comprises aportion of a conjugation linker;

x² is absent, is a nonfunctional or hydrophobic amino acid residue, orcomprises a portion of a conjugation linker;

x³ is absent, is basic or polar amino acid residue, or comprises aportion of a conjugation linker;

x⁴ is absent, is a basic or polar amino acid residue, or comprises aportion of a conjugation linker;

x⁵ is absent or is a nonfunctional, hydrophobic, or polar residue (e.g.pE, Q, Cit, L, V, G, H or P), or comprises a portion of a conjugationlinker;

x⁶ is absent or is a polar or basic residue (e.g. K or Cit, R, NMeArg,or hArg);

x⁷ is a nonfunctional or hydrophobic residue (e.g. P, Oic, or G);

x⁸ is a basic or polar residue (e.g. Q, Cit, R, NMeArg, or hArg);

x⁹ is a nonfunctional or hydrophobic residue (e.g. V, I, or L preferred,NMeLeu or Cha);

x¹⁰ is a nonfunctional, polar, or hydrophobic residue, or comprises aportion of a conjugation linker (e.g. S, F, and 4F-Phe);

x¹¹ is a nonfunctional, polar, basic, or hydrophobic residue, orcomprises a portion of a conjugation linker;

x¹² is a nonfunctional, hydrophobic, polar, or basic residue;

x¹³ is a nonfunctional or aromatic residue (e.g. G);

x¹⁴ is a nonfunctional or hydrophilic residue (e.g. P and Oic);

x¹⁵ is a nonfunctional, polar, or hydrophobic residue (e.g. aliphatic,aromatic, hydrophobic residues);

x¹⁶ is a nonfunctional or a hydrophobic residue (e.g. F, 4I-Phe,4Cl-Phe, Bip, P, Oic); and

x¹⁷ is absent or is a hydrophobic residue (e.g. aromatic residues).

The amino acid residues in the above formula can be D- or L-amino acids,α- or β-amino acids, non-canonical amino acids or D or L- or α- orβ-forms of the non-canonical amino acids.

The term “amino acid” or “residue” should be understood to mean acompound containing an amino group (NH₂), a carboxylic acid group(COOH), and any of various side groups, that have the basic formulaNH₂CHRCOOH, and that link together by peptide bonds to form proteins.Amino acids may, for example, be acidic, basic, aromatic, polar orderivatized. Non-standard amino acids may be referred to as“non-canonical” amino acids. Amino acids are naturally found in the α-and L-form, however, β- and D-form amino acids can also be prepared.(Araghi, R. R. et al., Amino Acids 41:733-742, 2011) In α-amino acids,the carboxylic acid group and the amino group are bonded to the samecarbon center. In β-amino acids, the amino group is bonded to the 13carbon which is found in most amino acids except glycine. In general,β-amino acids are rarely observed in nature relative to the dominantα-form.

A one-letter abbreviation system is frequently applied to designate theidentities of the twenty “canonical” amino acid residues generallyincorporated into naturally occurring peptides and proteins (Table 1).Such one-letter abbreviations are entirely interchangeable in meaningwith three-letter abbreviations, or non-abbreviated amino acid names.Within the one-letter abbreviation system used herein, an upper caseletter indicates an L-amino acid, and a lower case letter indicates aD-amino acid. For example, the abbreviation “R” designates L-arginineand the abbreviation “r” or “[1]” designates D-arginine.

TABLE 1 One-Letter Abbreviations for the Canonical Amino Acids (Threeletter abbreviations are in parentheses.) Alanine (Ala) A Glutamine(Gln) Q Leucine (Leu) L Serine (Ser) S Arginine (Arg) R Glutamic Acid(Glu) E Lysine (Lys) K Threonine (Thr) T Asparagine (Asn) N Glycine(Gly) G Methionine (Met) M Tryptophan (Trp) W Aspartic Acid (Asp) DHistidine (His) H Phenylalanine (Phe) F Tyrosine (Tyr) Y Cysteine (Cys)C Isoleucine (Ile) I Proline (Pro) P Valine (Val) V

An amino acid substitution in an amino acid sequence can be designatedherein with a one-letter abbreviation for the amino acid residue in aparticular position, followed by the numerical amino acid positionrelative to a native sequence of interest, which is then followed by theone-letter symbol for the amino acid residue substituted in. Forexample, “T30D” symbolizes a substitution of a threonine residue by anaspartate residue at amino acid position 30, relative to the nativesequence of interest.

Amino acid residues are commonly categorized according to differentchemical and/or physical characteristics. The term “acidic amino acidresidue” refers to amino acid residues in D- or L-form having sidechains comprising acidic groups. Exemplary acidic residues includeaspartic acid and glutamic acid residues. The term “alkyl amino acidresidue” refers to amino acid residues in D- or L-form having C₁₋₆ alkylside chains which may be linear, branched, or cyclized, including to theamino acid amine as in proline, wherein the C₁₋₆ alkyl is substituted by0, 1, 2 or 3 substituents selected from C₁₋₄haloalkyl, halo, cyano,nitro, —C(═O)R^(b), —C(═O)OR^(a), —C(═O)NR^(a)R^(a),—C(═NR^(a))NR^(a)R^(a), —NR^(a)C(═NR^(a))NR^(a)R^(a), —OR^(a),—OC(═O)R^(b), —OC(═O)NR^(a)R^(a), —OC₂₋₆alkylNR^(a)R^(a),—OC₂₋₆alkylOR^(a), —SR^(a), —S(═O)R^(b), —S(═O)₂R^(b),—S(═O)₂NR^(a)R^(a), —NR^(a)R^(a), —N(R^(a))C(═O)R^(b),—N(R^(a))C(═O)OR^(b), —N(R^(a))C(═O)NR^(a)R^(a),—N(R^(a))C(═NR^(a))NR^(a)R^(a), —N(R^(a))S(═O)₂R^(b),—N(R^(a))S(═O)₂NR^(a)R^(a), —NR^(a)C₂₋₆alkylNR^(a)R^(a) and—NR^(a)C₂₋₆alkylOR^(a); wherein R^(a) is independently, at eachinstance, H or R^(b); and R^(b) is independently, at each instanceC₁₋₆alkyl substituted by 0, 1, 2 or 3 substituents selected from halo,C₁₋₄ alkyl, C₁₋₃haloalkyl, —OC₁₋₄alkyl, —NH₂, —NHC₁₋₄alkyl, and—N(C₁₋₄alkyl)C₁₋₄alkyl; or any protonated form thereof, includingalanine, valine, leucine, isoleucine, proline, serine, threonine,lysine, arginine, histidine, aspartate, glutamate, asparagine,glutamine, cysteine, methionine, hydroxyproline, cyclohexylalanine,norleucine, norvaline, 2-aminobutyric acid, but which residues do notcontain an aryl or aromatic group.

The term “aromatic amino acid residue” refers to amino acid residues inD- or L-form having side chains comprising aromatic groups. Exemplaryaromatic residues include tryptophan, tyrosine, 3-(1-naphthyl)alanine,histidine, or phenylalanine residues. The term “basic amino acidresidue” refers to amino acid residues in D- or L-form having sidechains comprising basic groups. Exemplary basic amino acid residuesinclude histidine, lysine, homolysine, ornithine, arginine,N-methyl-arginine, ω-aminoarginine, ω-methyl-arginine,1-methyl-histidine, 3-methyl-histidine, and homoarginine (hR) residues.The term “hydrophilic amino acid residue” or “polar amino acid residue”refers to amino acid residues in D- or L-form having side chainscomprising polar groups. Exemplary hydrophilic or polar residues includecysteine, serine, threonine, histidine, lysine, asparagine, aspartate,glutamate, glutamine, and citrulline (Cit) residues. The term“hydrophobic amino acid residue” or “lipophilic amino acid residue”refers to amino acid residues in D- or L-form having sidechainscomprising uncharged, aliphatic or aromatic groups. Exemplary lipophilicsidechains include phenylalanine, isoleucine, leucine, norleucine,methionine, valine, tryptophan, and tyrosine. Alanine (A) isamphiphilic—it is capable of acting as a hydrophilic, or lipophilic(i.e., hydrophobic), residue. Alanine, therefore, is included within thedefinition of both “lipophilic” (i.e., “hydrophobic”) residue and“hydrophilic” residue. The term “nonfunctional” or “neutral” amino acidresidue refers to amino acid residues in D- or L-form having side chainsthat lack acidic, basic, or aromatic groups. Exemplary neutral aminoacid residues include methionine, glycine, alanine, valine, isoleucine,leucine, and norleucine.

The term “non-canonical” or “unnatural” amino acids refers to amino acidresidues in D- or L-form that are not among the 20 canonical amino acidsgenerally incorporated into naturally occurring proteins. Examples ofnon-canonical amino acids can be found in FIG. 1 and Table 2.Non-canonical amino acid residues can be incorporated into a peptide byemploying known techniques of protein engineering that use recombinantlyexpressing cells. See, e.g., Link et al., Non-canonical amino acids inprotein engineering, Current Opinion in Biotechnology, 14(6):603-609,2003. Additional examples of non-canonical amino acids include, but arenot limited to, for example, β-amino acids, homoamino acids, cyclicamino acids and amino acids with derivatized side chains. Additionalexamples can include (in the L-form or D-form) β-alanine,β-aminopropionic acid, piperidinic acid, aminocaprioic acid,aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic acid,N^(α)-ethylglycine, N^(α)-ethylaspargine, hydroxylysine,allo-hydroxylysine, isodesmosine, allo-isoleucine, ω-methylarginine,N^(α)-methylglycine, N^(α)-methylisoleucine, N^(α)-methylvaline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N^(α)-acetylserine, N^(α)-formylmethionine,3-methylhistidine, 5-hydroxylysine, and other similar amino acids, andthose listed in Table 2 below, and derivatized forms of any of these asdescribed herein. Table 2 and FIG. 1 contain some exemplarynon-canonical amino acid residues that may be incorporated into themodified apelin polypeptides of the invention as well as the associatedabbreviations as typically used herein. However, the ordinary-skilledartisan will understand that different abbreviations and nomenclaturesmay be applicable to the same substance and appear interchangeablyherein. Some amino acid sequences, as recited herein may include “{H},”“{H2},” or “{Hydrogen}” at the N-terminus, all of which represent anN-terminal amino group, and/or may include “-{Free Acid}” or {COOH} atthe C-terminus, both of which represent a C-terminal carboxy group. Somesequences include an “Ac,” “Acetyl,” or “Acetyl-NH” at the N-terminus,which indicates acetylation at the N-terminus (e.g. acetate oracetamide). As used herein, the term “bromoacetyl” refers tobromoacetate or bromoacetamide.

In the event an abbreviation listed in Table 2 differs from anotherabbreviation for the same substance disclosed elsewhere herein, bothabbreviations are understood to be applicable. The amino acids listed inTable 2 can be in the L-form or D-form, unless otherwise noted.

TABLE 2 Examples of Non-Canonical Amino Acids for Use in PeptideSequences AMINO ACID ABBREVIATIONS 1’N-methyltryptophan 1’NMeW1-aminocyclohexanecarboxylic acid 1-Ach 3-(1-naphthyl)alanine 1-Nal;1Nal 2-aminobutyric acid 2-Abu 2-chloro-L-phenylalanine 2-Cl—F3-(2-naphthyl)alanine 2-Nal; 2Nal 2-pyridinylalanine 2Pal2-amino-3-guanidinopropanoic acid 3G-Dpr 3-pyridinylalanine 3Pal4-amino-phenylalanine (also known as para- 4AmP; 4-AminoF; 4-Amino-Pheaminophenylalanine) 4-amidino-phenylalanine 4AmPhe2-amino-2-(1-carbamimidoylpiperidin-4-yl)acetic acid 4AmPig4-benzoyl-L-phenylalanine 4-Bz—F 4-trifluoromethyl-L-phenylalanine4CF3—F 4-chloro-L-phenylalanine 4-Cl—F 4-carboxyphenylalanine 4CO2—F4-fluoro-L-phenylalanine 4-F—F 4-guanidino proline 4GuaPr4-methyl-L-phenylalanine 4-Me—F 4-pyridinylalanine 4Pal4-amino-1-piperidine-4-carboxylic acid 4Pip 4-piperidinylalanine 4PipA4-tert-butyl-L-phenylalanine 4tBu—F α-aminoadipic acid Aad aminobutyricacid Abu α-carboxy-4-aminobutyric acid AC4Abu; γGlu Acetylarginineacetylarg Acetamidomethyl Acm 1-aminocyclopentanecarboxylic acid Acp[2-(2-Amino-ethoxy)-ethoxy]-acetic acid Aeea3-amino-6-hydroxy-2-piperidone Ahp 6-aminohexanoic acid Ahx; εAhxα-amino-isobutyric acid Aib 2-aminoindane-2-carboxylic acid Aicα-methylphenyalanine AMeF α-methyl-leucine aMeLeu α-methyl-lysine aMeLysα-methyl-ornithine aMeOrn α-methyl-proline aMePro α-metyl-serine aMeSAminophenylalanine Aminophe; Amino-Phe 3-amino-2-naphthoic acid Anc2-aminotetraline-2-carboxylic acid Atc 3-(1,2,3-triazol-4-yl)Alanine Atz3-(1-(O-(aminoethyl)-O’-(ethylene)- Atz(20 kDa PEG)ethyleneglycol450avg)-1,2,3-triazol-4-yl)Alanine3-(1-(O-(aminoethyl)-O’-(ethylene)- Atz(amino-PEG10)decaethyleneglycol)-1,2,3-triazol-4-yl)Alanine(S)-2-amino-3-(1-(1-hydroxy-5-oxo-9,12,15,18,21,24,Atz(PEG11-((2-hydroxyethyl)27,30,33,36,39-undecaoxa-3-thia-6-azahentetracontan- thio)acetamide)41-yl)-1H-1,2,3-triazol-4-yl)propanoic acid(S)-2-amino-3-(1-(2-oxo-6,9,12,15,18,21,24,27,30,Atz(PEG11-(acetamidomethyl)33,36-undecaoxa-3-azaoctatriacontan-38-yl)-1H-1,2,3-triazol-4-yl)propanoic acid(S)-2-amino-3-(1-(1-bromo-2-oxo-6,9,12,15,18,21,Atz(PEG11-bromoacetamide)24,27,30,33,36-undecaoxa-3-azaoctatriacontan-38-yl)-1H-1,2,3-triazol-4-yl)propanoic acid(S)-2-amino-3-(1-(1-bromo-2-oxo-6,9,12-trioxa-3-Atz(PEG3-bromoacetamide)azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)propanoic acidβ-4-chloro-L-phenylalanine B4ClF β-alanine bAlaβ-4-chloro-L-homophenylalanine Bh4ClF β-homoarginine BhArgβ-homoasparagine BhAsn β-homocyclohexylalanine BhCha β-homoglutamineBhGln β-homoleucine BhLeu β-homolysine BhLys β-homonorleucine BhNleβ-homophenylalanine BhPhe β-homoproline BhPro β-homoserine BhSer β-homoTic BhTic β-homotryptophan BhTrp 4,4′-biphenylalanine;4-phenyl-phenylalanine; or Bip; 4Bip biphenylalanineβ,β-diphenyl-alanine BiPhA β-leucine BLeu β-lysine BLys β-norleucineBNle β-ornithine BOrn β-phenylalanine BPhe L-cysteine involved in asidechain cyclization C1 Cyclohexylalanine Cha Cyclohexylglycine ChgCitrulline Cit p-carboxyl-phenylalanine Cpa Cyclopentylglycine CPGL-Aspartate involved in a sidechain cyclization D13-(1-naphthyl)-D-alanine D-1Nal 3-(2-naphthyl)-D-alanine D-2Nal4-chloro-D-phenylalanine D-4ClF 4-Iodo-D-phenylalanine D-4IF α,γ-diaminobutyric acid Dab diaminopropionic acid Dapβ-homo-D-phenylalanine D-BhPhe(R)-3-([1,1′-biphenyl]-4-yl)-2-aminopropanoic acid D-Bip2,4-diaminobutyric acid Dbu 2-amino-2-ethylbutanoic acid Deg3,4-dichloro-L-phenylalanine DiCl—F(R)-2-amino-2-(2,3-dihydro-1H-inden-2-yl)acetic acid D-Igl3,4-dimethoxy-L-phenylalanine DiMeO—F 3,3-diphenylalanine DipD-Ornithine D-ORN α, β-diaminopropionoic acid (or 2,3-diaminopropionicDpr acid D-Norleucine ψ(CH2NH)-reduced amide bond DrNle D-Serineψ(CH2NH)-reduced amide bond DrSer(R)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid D-Tic(R)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid D-Tiq L-Glutamateinvolved in a sidechain cyclization E1 4-guanidino phenylalanine Gufhomo-L-Arginine hArg Homoarginine hArg; hR Homocitrulline hCitHomoleucine hLeu; hL Homolysine hLys; hK; homoLys HomophenylalaninehPhe; homoPhe Homoglutamine hQ1,2,3,4-tetrahydroisoquinoline-7-hydroxy-3-carboxylic Hydroxyl-Tic acid4-hydroxyproline (or hydroxyproline) Hyp indoline-2-carboxylic acid Idc2-indanylglycine (or indanylglycine) IgI(S)-2-amino-2-(2,3-dihydro-1H-inden-2-yl)acetic acid Iglpiperidine-4-carboxylic acid Inp Iodotyrosine I-Tyr(S)-2-amino-6-(pent-4-ynamido)hexanoic acid K(4-Pen)(S)-6-((S)-2-acetamidopent-4-ynamido)-2- K(Ac-Pra) aminohexanoic acidN-ε-biotinyl-L-lysine K(Biotin)(S)-2,2′,2″-(10-(2-((5-amino-5-carboxypentyl)amino)- K(DOTA)2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7- triyl)triacetic acid(S)-2-amino-6-(3-(1-(1-bromo-2-oxo-6,9,12,15,18,21,K(ethyl-triazole-PEG11-24,27,30,33,36-undecaoxa-3-azaoctatriacontan-38-yl)- bromoacetamide)1H-1,2,3-triazol-4-yl)propanamido)hexanoic acid N-ε-dimethyl lysineK(Me2) Nε-(O-(aminoethyl)-O′-(2-propanoyl)- K(NPeg11)undecaethyleneglycol)-LysineNε-(O-(aminoethyl)-O′-(2-propanoyl)-(ethyleneglycol) K(NPeg27) 27-LysineL-Lysine involved in a sidechain cyclization K1 4-methyl-phenylalanineMePhe methionine oxide Met[O] methionine sulfone Met[O]2N^(α)-methylhomocitrulline N a-MeHoCit N^(α)-methylornithine N a-MeOrn;NMeOrn 2-((4-chlorobenzyl)amino)acetic acid N4ClFG N^(α)-methylleucineNa—MeL; NMeL; NMeLeu; NMe-Leu N^(α)-[(CH2)3NHCH(NH)NH2] substitutedglycine N-Arg Nε-ethyl-lysine N-eEt-K Nε-isopropyl-lysine N-eIPr-KNε-methyl-lysine N-eMe—K N-α-(2-hydroxyethyl)-glycine NhSerG nipecoticacid Nip Nitrophenylalanine nitrophe norleucine Nle2-((4-aminobutyl)amino)acetic acid NLysG(S)-3-(4-chlorophenyl)-2-(methylamino)propanoic acid NMe4ClF(S)-5-guanidino-2-(methylamino)pentanoic acid; N^(α)- NMeArgmethylarginine (S)-3-cyclohexyl-2-(methylamino)propanoic acid NMeChaN^(α)-methylcitrulline NMeCit (S)-5-amino-2-(methylamino)-5-oxopentanoicacid NMeGln (S)-6-guanidino-2-(methylamino)hexanoic acid NMehArg(S)-3-(1H-imidazol-4-yl)-2-(methylamino)propanoic NMeHis acidN^(α)-methylhomolysine NMeHoK (S)-4-methyl-2-(methylamino)pentanoic acidNMeLeu (S)-6-amino-2-(methylamino)hexanoic acid; Nα- NMeLys methyllysine(S)-2-(methylamino)hexanoic acid; N^(α)- NMeNle methylnorleucine(S)-2-(methylamino)-3-phenylpropanoic acid; N^(α)- NMePhemethylphenylalanine N^(α)-methylglutamine NMeQ(S)-3-hydroxy-2-(methylamino)propanoic acid NMeSer(2S)-3-hydroxy-2-(methylamino)butanoic acid; N^(α)- NMeThrmethylthreonine (S)-3-methyl-2-(methylamino)butanoic acid; N^(α)- NMeValmethylvaline 2-((3-aminopropyl)amino)acetic acid NOrnG2-(piperidin-4-ylamino)acetic acid NPipG 2-(propylamino)acetic acid NPrGnorvaline Nva norvaline Nva or Nyl Ornithine O (S)-octylglycine OctylGoctahydroindole-2-carboxylic acid Oic O-methyltyrosine Ome-Tyr OrnithineOrn pyroglutamic acid pGlu; PE; pE Phenylglycine Phg pipecolic acid Pippara-iodophenylalanine (or 4-iodophenylalanine) pI-Phe PropargylglycinePra L-phosphoserine pS ω-N-methylarginine R(Me) Arg ψ(CH2NH)-reducedamide bond rArg Cyclohexylalanine ψ(CH2NH)-reduced amide bond rChahomoArginineψ(CH2NH)-reduced amide bond rhArg Histidine ψ(CH2NH)-reducedamide bond rHis Lysine ψ(CH2NH)-reduced amide bond rLys Norleucineψ(CH2NH)-reduced amide bond rNle Orn ψ(CH2NH)-reduced amide bond rOrnPhenylalanine ψ(CH2NH)-reduced amide bond rPhe Serine ψ(CH2NH)-reducedamide bond rSer Sarcosine Sar symmetrical N’-ω-dimethyl arginine SDMA3-thienylalanine Thi thiazolidine-4-carboxylic acid Thz1,2,3,4-tetrahydroisoquinoline Tic1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid Tiq(S)-tert-butylglycine Tle 1,2,3,4-tetrahydronorharman-3-carboxylic acidTpi

The modified apelin polypeptides of the invention can be from about 10amino acids to about 80 amino acids in length, from about 13 amino acidsto about 40 amino acids in length, or from about 15 amino acids to about25 amino acids in length. In certain embodiments, the modified apelinpolypeptides are about 12 amino acids in length. In other embodiments,the modified apelin polypeptides are about 13 amino acids in length. Inone particular embodiment, the modified apelin polypeptide is about 15amino acids in length. In another particular embodiment, the modifiedapelin polypeptide is about 17 amino acids in length. In someembodiments, the modified apelin polypeptide is about 36 amino acids inlength.

In certain embodiments, the modified apelin polypeptides of theinvention have at least one non-canonical amino acid. In otherembodiments, the modified apelin polypeptides have at least two, atleast three, at least four, at least five, at least six, at least seven,at least eight, or at least nine non-canonical amino acids. The modifiedapelin polypeptides can have at least 25% of the total amino acids inthe peptide as non-canonical amino acids. For instance, in someembodiments, at least 30% of the amino acids in the polypeptide arenon-canonical amino acids. In one embodiment, the modified apelinpolypeptides of the invention have at least 50% of the amino acids inthe polypeptide as non-canonical amino acids. In another embodiment, atleast 60% of the amino acids in the modified apelin polypeptide arenon-canonical amino acids. In still another embodiment, at least 70% ofthe amino acids in the modified apelin polypeptide are non-canonicalamino acids. The non-canonical amino acids can be any of those disclosedherein, including those listed in Table 2.

In some embodiments, the modified apelin polypeptides of the inventioncomprise the amino acid sequence:

X₁ X₂ X₃X₄ X₅ X₆ X₇ X₈ X₉X₁₀GX₁₁X₁₂ X₁₃ X₁₄ (SEQ ID NO: 717), wherein:

X₁ is R, E, [hArg], or absent;

X₂ is [r], R, E, [hArg], or absent;

X₃ is Q, [q], or [BLeu];

X₄ is [hArg], [NMeArg], R, E, or [r];

X₅ is P or [aMePro];

X₆ is R, E, [r], [hArg] or [NMeArg];

X₇ is L, [aMeLeu], [BLeu], [NMeLeu] or [Cha];

X₈ is S, [BhSer], or [NhSerG];

X₉ is H or Y;

X₁₀ is K or [NLysG];

X₁₁ is P, [Oic], [aMePro], or [Pip];

X₁₂ is [Nle], [rNle], or [pI-Phe];

X₁₃ is P, [BhPro], [aMePro], or [Aib]; and

X₁₄ is F, [D-BhPhe], [4-Cl-F], [D-4ClF], or [D-Bip].

In some such embodiments, X₇ is [NMeLeu], X₁₂ is [pI-Phe], and X₁₄ is[D-Bip]. In related embodiments, X₁ is [hArg], X₂ is [hArg], X₃ is Q, X₄is [hArg], and X₅ is P. In certain embodiments, X₆ and X₇ are,respectively, [NMeArg] and [aMeLeu], [hArg] and [BLeu], or [hArg] and[aMeLeu]. In other particular embodiments, X₁₃ is [BhPro], [aMePro], or[Aib] and X₁₄ is [D-BhPhe] or [4-Cl-F]. The modified apelin polypeptidesof the invention can comprise a sequence selected from SEQ ID NOs: 8-11,16, 17, 31, 32, 45, 53, 60, 68, 69-71, 92, 112, 114, 119, 120, 221, 228,237, 263, 286, 287, 362, 373, 376, 379, 382, 388, 412, 416, 460, 468,482, 483, 485, 491, 498, 499, 500, 502, 505, 514, 519, 526, 531, 534,544, 552, 554, 560, and 571. In some embodiments, the modified apelinpolypeptide comprises the amino acid sequence selected from SEQ ID NOs:19, 20, 22-24, 29, 30, 33, 34, 37, 46, 47, 50-52, 55, 56, 97-105, 107,109, 111, 115, 117, 118, 127, 150, 154, 156, 157, 176-181, 183-185,211-232, 236, 241, 242, 261-267, 270, 274, 275, 278-281, 284, 349, 540,and 698.

The modified apelin polypeptides of the invention may be acetylated atthe N-terminus. For instance, in one embodiment, the modified apelinpolypeptides comprise the amino acid sequence:

Ac-X₁ X₂ X₃ X₄X₅ X₆X₇ X₈X₉ X₁₀X₁₁X₁₂ X₁₃ X₁₄ X₁₅ X₁₆ X₁₇ (SEQ ID NO:718), wherein: X₁ is O or [BhLys]; X₂ is F, [BhPhe] or [Bphe]; X₃ is[hArg], [NmeArg] or [BhArg]; X₄ is [hArg], [NmeArg] or [BhArg]; X₈ is Q,[BhGln], [BhAsn] or [BhLeu]; X₆ is [hArg], [NmeArg] or [BhArg]; X₇ is P,[Sar], [Aib], [BhPro] or [Pip]; X₈ is [hArg], [NmeArg] or [BhArg]; X₉ is[Cha] or [BhLeu]; X₁₀ is S, [BhSer], [Sar] or [bAla]; X₁₁ is H, [NmeVal]or [bAla]; X₁₂ is K, [NmeLys], [BhLys], [Blys] or [bAla]; X₁₃ is G,[Sar], [Aib] or [bAla]; X₁₄ is [Oic], [Aib], [Sar], [bAla], [BhPro] or[Pip]; X₁₅ is [Nle] or [bAla]; X₁₆ is P, [Sar], [Aib], [BhPro], [bAla],[Pip], [D-1Nal] or [D-2Nal]; and X₁₇ is [4-Cl-F], [Bh-Phe] or [Bphe]. Insuch embodiments, the modified apelin polypeptide may comprise an aminoacid sequence of any one of the peptides listed in Table 3 below.

TABLE 3 Exemplary Modified Apelin Polypeptides SEQ ID NO: SEQUENCE 668OF[hArg][hArg]Q[hArg]P[NMeArg][Cha]SHKG[Oic][Nle][1Nal] 669OF[NMeArg][hArg]Q[hArg]P[hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] 670OF[hArg][NMeArg]Q[hArg]P[hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] 671OF[hArg][hArg]Q[NMeArg]P[hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] 672OF[hArg][hArg]Q[NMeArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] 673OF[hArg][NMeArg]Q[NMeArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] 674[hArg]Q[hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] 675Q[hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] 676OF[hArg][hArg]Q[hArg][Sar][hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] 677OF[hArg][hArg]Q[hArg][Aib][hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] 678OF[hArg][hArg]Q[hArg]P[hArg][Cha][Sar]HKG[Oic][Nle]P[4-Cl—F] 679OF[hArg][hArg]Q[hArg]P[hArg][Cha]S[NMeVal]KG[Oic][Nle]P[4-Cl—F] 680OF[hArg][hArg]Q[hArg]P[hArg][Cha]SHK[Sar][Oic][Nle]P[4-Cl—F] 681OF[hArg][hArg]Q[hArg]P[hArg][Cha]SHK[Aib][Oic][Nle]P[4-Cl—F] 682OF[hArg][hArg]Q[hArg]P[hArg][Cha]SHKG[Aib][Nle]P[4-Cl—F] 683OF[hArg][hArg]Q[hArg]P[hArg][Cha]SHKG[Sar][Nle]P[4-Cl—F] 684OF[hArg][hArg]Q[hArg]P[hArg][Cha]SHKG[Oic][Nle][Sar][4-Cl—F] 685OF[hArg][hArg]Q[hArg]P[hArg][Cha]SHKG[Oic][Nle][Aib][4-Cl—F] 686O[BhPhe][hArg][hArg]Q[hArg]P[hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] 687O[BPhe][hArg][hArg]Q[hArg]P[hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] 688OF[BhArg][hArg]Q[hArg]P[hArg][Cha]SHKG[Oic][Nle]P[4lF] 689OF[hArg][BhArg]Q[hArg]P[hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] 690OF[hArg][hArg][BhGln][hArg]P[hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] 691OF[hArg][hArg][BhAsn][hArg]P[hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] 692OF[hArg][hArg][BhLeu][hArg]P[hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] 693OF[hArg][hArg]Q[BhArg]P[hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] 694OF[hArg][hArg]Q[hArg][BhPro][hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] 695OF[hArg][hArg]Q[hArg][Pip][hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] 696OF[hArg][hArg]Q[hArg]P[BhArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] 697OF[hArg][hArg]Q[hArg]P[hArg][BhLeu]SHKG[Oic][Nle]P[4-Cl—F] 698OF[hArg][hArg]Q[hArg]P[hArg][Cha][BhSer]HKG[Oic][Nle]P[4-Cl—F] 699OF[hArg][hArg]Q[hArg]P[hArg][Cha][bAla]HKG[Oic][Nle]P[4-Cl—F] 700OF[hArg][hArg]Q[hArg]P[hArg][Cha]S[bAla]KG[Oic][Nle]P[4-Cl—F] 701OF[hArg][hArg]Q[hArg]P[hArg][Cha]SH[BhLys]G[Oic][Nle]P[4-Cl—F] 702OF[hArg][hArg]Q[hArg]P[hArg][Cha]SH[BLys]G[Oic][Nle]P[4-Cl—F] 703OF[hArg][hArg]Q[hArg]P[hArg][Cha]SH[bAla]G[Oic][Nle]P[4-Cl—F] 704OF[hArg][hArg]Q[hArg]P[hArg][Cha]SHK[bAla][Oic][Nle]P[4-Cl—F] 705OF[hArg][hArg]Q[hArg]P[hArg][Cha]SHKG[bAla][Nle]P[4-Cl—F] 706OF[hArg][hArg]Q[hArg]P[hArg][Cha]SHKG[BhPro][Nle]P[4-Cl—F] 707OF[hArg][hArg]Q[hArg]P[hArg][Cha]SHKG[Pip][Nle]P[4-Cl—F] 708OF[hArg][hArg]Q[hArg]P[hArg][Cha]SHKG[Oic][bAla]P[4-Cl—F] 709OF[hArg][hArg]Q[hArg]P[hArg][Cha]SHKG[Oic][Nle][BhPro][4-Cl—F] 710OF[hArg][hArg]Q[hArg]P[hArg][Cha]SHKG[Oic][Nle][bAla][4-Cl—F] 711OF[hArg][hArg]Q[hArg]P[hArg][Cha]SHKG[Oic][Nle][Pip][4-Cl—F] 712OF[hArg][hArg]Q[hArg]P[hArg][Cha]SHKG[Oic][Nle][D-1Nal] 713OF[hArg][hArg]Q[hArg]P[hArg][Cha]SHKG[Oic][Nle][D-2Nal] 714OF[hArg][hArg]Q[hArg]P[hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F]

In other embodiments, acetylated modified apelin polypeptides of theinvention comprise the amino acid sequence: [z]X₁ X₂ X₃X₄ X₅ X₆X₇SHX₈G[Oic]X₉ X₁₀ X₁₁ (SEQ ID NO: 719), wherein: z is acetyl or absent;X₁ is acetyl, [r] or [hArg]; X₂ is [r], R or [hArg]; X₃ is Q or [q]; X₄is [hArg], R or [r]; X₅ is P or [Oic]; X₆ is[r], [hArg] or [NMeArg]; X₇is [NMeLeu] or [Cha]; X₈ is K or [NLysG]; X₉ is [pl-Phe] or [Nle]; X₁₀is P or [D-1Nal] or [Pip]; and X₁₁ is a D-amino acid, a β-amino acid, anon-canonical amino acid or the D- or β-form of the non-canonical aminoacid. In such embodiments, X₁₁ is [D-Bip], [4-CL-F], [D-4CLF], [TIC],[f] or is absent. Exemplary modified apelin polypeptides, includingacetylated modified apelin polypeptides, are listed in Table 4 below. Incertain embodiments, the modified apelin polypeptide may comprise anamino acid sequence of any one of the peptides listed in Table 4 below.

TABLE 4 Additional Exemplary Modified Apelin Polypeptides SEQ ID NO:SEQUENCE 7 RQRP[r][NMeLeu]SHKG[Oic][pIPhe]P[D-Bip] 8QrP[hArg][aMeLeu]SHKGP[Nle]P[4-Cl—F]{COOH} 9QR[aMePro][hArg][aMeLeu]SHKGP[Nle]P[4-Cl—F]{COOH} 10Q[NMehArg]P[NMeArg][aMeLeu]SHKGP[Nle]P[4-Cl—F]{COOH} 11QRP[NMeArg][Cha]SHKG[Oic][Nle][Aib][4-Cl—F]{COOH} 12{H2}KFRRQRPRLSHKGPMP{COOH} 13 {H2}CMPLHSRVPFP{COOH} 14{H2}KLRKHN[Abu]LQRR[Abu]MPLHSRVPFP{COOH} 15{H2}KLRKHNCLQRRCMPLHSRVPFP{COOH} 16Acetyl-[hArg][r]Q[hArg]P[r][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip] {COOH} 17Acetyl-RQRP[hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F]{COOH} 18Acetyl-[r][hArg]Q[hArg]P[r][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip] {COOH} 19Acetyl-[hArg][hArg][q][hArg]P[r][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip] {COOH}20 Acetyl-[hArg][hArg]Q[r]P[r][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip] {COOH}21 Acetyl-[hArg][hArg]Q[hArg][Oic][r][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 22 Acetyl-[hArg][hArg]Q[r]P[NMeArg][Cha]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 23 Acetyl-[hArg]Q[r]P[NMeArg][Cha]SHKG[Oic][Nle]P[4-Cl—F]{COOH}24 Acetyl-[hArg]Q[r]P[NMeArg][Cha]SHKG[Oic][Nle]P[D-4ClF]{COOH} 25Acetyl-[hArg]Q[r]P[NMeArg][Cha]SHKG[Oic][Nle][D-1Nal]{COOH} 26Acetyl-Q[r]P[NMeArg][Cha]SHKG[Oic][Nle]P[Tic]{COOH} 27Acetyl-[q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[f]{COOH} 28Acetyl-[hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[f]{COOH} 29Acetyl-OF[hArg][hArg][BLeu][hArg]P[hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F]{COOH} 30Acetyl-OF[hArg][hArg]Q[hArg]P[hArg][BLeu]SHKG[Oic][Nle]P[4-Cl—F] {COOH}31 {Acetyl}OF[hArg][hArg]Q[hArg]P[hArg][Cha][NhSerG]HKG[Oic][Nle]P[4-Cl—F]{COOH} 32{Acetyl}OF[hArg][hArg]Q[hArg]P[hArg][Cha]SH[NLysG]G[Oic][Nle]P[4-Cl—F]{COOH} 33{Acetyl}OF[hArg][hArg]Q[hArg][aMePro][hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F]{COOH} 34{Acetyl}OF[hArg][hArg]Q[hArg]P[hArg][aMeLeu]SHKG[Oic][Nle]P[4-Cl—F]{COOH} 35{Acetyl}OF[hArg][hArg]Q[hArg]P[hArg][Cha][aMeS]HKG[Oic][Nle]P[4-Cl—F]{COOH} 36{Acetyl}OF[hArg][hArg]Q[hArg]P[hArg][Cha]SH[aMeOrn]G[Oic][Nle]P[4-Cl—F]{COOH} 37{Acetyl}OF[hArg][hArg]Q[hArg]P[hArg][Cha]SHKG[Oic][Nle][aMePro][4-Cl—F]{COOH} 38{Acetyl}OF[hArg][hArg]Q[rhArg]P[hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] {COOH}39 {Acetyl}OF[hArg][hArg]Q[hArg]P[rhArg][Cha]SHKG[Oic][Nle]P[4-Cl—F]{COOH} 40{Acetyl}OF[hArg][hArg]Q[hArg]P[hArg][Cha]S[rHis]KG[Oic][Nle]P[4-Cl—F]{COOH} 41{Acetyl}OF[hArg][hArg]Q[hArg]P[hArg][Cha]SH[rLys]G[Oic][Nle]P[4-Cl—F]{COOH} 42{Acetyl}OF[hArg][hArg]Q[hArg]P[hArg][rCha]SHKG[Oic][Nle]P[4-Cl— F]{COOH}43 {Acetyl}OF[hArg][hArg]Q[hArg]P[hArg][Cha][rSer]HKG[Oic][Nle]P[4-Cl—F]{COOH} 44{Acetyl}OF[hArg][hArg]Q[hArg]P[hArg][Cha][DrSer]HKG[Oic][Nle]P[4-Cl—F]{COOH} 45{Acetyl}OF[hArg][hArg]Q[hArg]P[hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] {COOH}46 {Acetyl-NH}OF[hArg][hArg]QRP[hArg][Cha]SHKGP[pI-Phe]P[D-Bip] {COOH}47 {Acetyl-NH}OF[hArg][hArg]QRP[hArg][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 48 {Acetyl-NH}OF[hArg][hArg]QRP[hArg][Cha]SHKGP[Nle][4-Cl—F]{COOH} 49 {Acetyl-NH}OF[hArg][hArg]QRP[hArg][Cha]SHKGP[Nle][2-Nal]{COOH}50 {Acetyl-NH}rQ[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 51{Acetyl-NH}RQRP[hArg][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 52{Acetyl-NH}rQRP[hArg][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 53{Acetyl-NH}Q[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 54{Acetyl-NH}QrP[NMeArg][Cha]SHKG[Oic][Nle][D-1Nal]{COOH} 55{Acetyl-NH}q[hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[D-4ClF]{COOH} 56{Acetyl-NH}[hArg][hArg]Q[hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[4-Cl—F]{COOH} 57{Acetyl-NH}OF[hArg][hArg]Q[hArg]P[NMeArg][Cha]SHKG[Oic][Nle][4-Cl—F]{COOH} 58{Acetyl-NH}OF[hArg][hArg]Q[hArg][Oic]r[Cha]SHKG[Oic][Nle][4-Cl— F]{COOH}59 {Acetyl-NH}OF[hArg][hArg]Q[hArg]Pr[NMeLeu]SHKG[Oic][Nle][4-Cl—F]{COOH} 60 {Acetyl-NH} [hArg] Q [hArg] P [NMeArg] [Cha] S H K G [Oic][Nle] P [4- Cl—F]{COOH} 61 {Acetyl-NH} Q [hArg] P [NMeArg] [Cha] S H K G[Oic] [Nle] P [4-Cl—F] {COOH} 62 {Acetyl-NH} R Q R P R L S H K G P [Nle]P [D-Bip]{COOH} 63 {Acetyl-NH} R Q R P R [Cha] S H K G P [Nle] P[4-Cl—F]{COOH} 64 {Acetyl-NH} R Q R P R [NMeLeu] S H K G P [Nle] P[4-Cl—F] {COOH} 65 {Acetyl-NH} R Q R P [NMeArg] L S H K G P [Nle] P[4-Cl—F] {COOH} 66 {Acetyl-NH} R Q R P [NMeArg] [Cha] S H K G P [Nle] P[4-Cl—F] {COOH} 67 {Acetyl-NH} R Q R P [NMeArg] [NMeLeu] S H K G P [Nle]P [4-Cl—F] {COOH} 68 {Acetyl-NH} Q R P [NMeArg] L S H K G P [Nle] P[4-Cl—F] {COOH} 69 {Acetyl-NH} Q R [aMePro] [hArg] [aMeLeu] S H K G P[Nle] P [4-Cl—F] {COOH} 70 {Acetyl-NH} Q r P [hArg] [aMeLeu] S H K G P[Nle] P [4-Cl—F] {COOH} 71 {Acetyl-NH} Q [hArg] P [NMeArg] [aMeLeu] S HK G P [Nle] P [4-Cl—F] {COOH} 72 {Acetyl-NH} Q R [NMeArg] L S H K G P[Nle] P [4-Cl—F] {COOH} 73 {Acetyl-NH} Q R [NMehArg] L S H K G P [Nle] P[4-Cl—F] {COOH} 74 {Acetyl-NH} Q [hArg] [NMehArg] L S H K G P [Nle] P[4-Cl—F] {COOH} 75 {Acetyl-NH} E r Q [hArg] P r [NMeLeu] S H K G [Oic][pI-Phe] P [D-Bip] {COOH} 76 {Acetyl-NH} [hArg] E Q [hArg] P r [NMeLeu]S H K G [Oic] [pI-Phe] P [D- Bip] {COOH} 77 {Acetyl-NH} [hArg] r Q E P r[NMeLeu] S H K G [Oic] [pI-Phe] P [D-Bip] {COOH} 78 {Acetyl-NH} [hArg] rQ [hArg] E r [NMeLeu] S H K G [Oic] [pI-Phe] P [D- Bip] {COOH} 79{Acetyl-NH} [hArg] r Q [hArg] P E [NMeLeu] S H K G [Oic] [pI-Phe] P [D-Bip] {COOH} 80 {Acetyl-NH} [hArg] r Q [hArg] P r E S H K G [Oic][pI-Phe] P [D-Bip] {COOH} 81 {Acetyl-NH} [hArg] r Q [hArg] P r [NMeLeu]E H K G [Oic] [pI-Phe] P [D- Bip] {COOH} 82 {Acetyl-NH} [hArg] r Q[hArg] P r [NMeLeu] S E K G [Oic] [pI-Phe] P [D- Bip] {COOH} 83{Acetyl-NH} [hArg] r Q [hArg] P r [NMeLeu] S H E G [Oic] [pI-Phe] P [D-Bip] {COOH} 84 {Acetyl-NH} [hArg] r Q [hArg] P r [NMeLeu] S H K E [Oic][pI-Phe] P [D- Bip] {COOH} 85 {Acetyl-NH} [hArg] r Q [hArg] P r [NMeLeu]S H K G E [pI-Phe] P [D-Bip] {COOH} 86 {Acetyl-NH} [hArg] r Q [hArg] P r[NMeLeu] S H K G [Oic] E P [D-Bip] {COOH} 87 {Acetyl-NH} [hArg] r Q[hArg] P r [NMeLeu] S H K G [Oic] [pI-Phe] E [D- Bip] {COOH} 88{Acetyl-NH} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] L S H K G P [Nle] P[4-Cl—F] {COOH} 89 {Acetyl-NH} S D F Y K R L I N K A K [Aeea] [Aeea] Q RP [NMeArg] L S H K G P [Nle] P [4-Cl—F] {COOH} 90 {Acetyl-NH} R L I E DI C L P R W G C L W E D D [Aeea] [Aeea] Q R P [NMeArg] L S H K G P [Nle]P [4-Cl—F] {COOH} 91 {Acetyl-NH} R L I E D I C L P R W G C L W [Aeea][Aeea] Q R P [NMeArg] L S H K G P [Nle] P [4-Cl—F] {COOH} 92 {Acetyl-NH}Q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl—F] {COOH} 93{Acetyl-NH} q R P [NMeArg] [Cha] S H K G [aMePro] [Nle] [Aib] [4-Cl—F]{COOH} 94 {Acetyl-NH} S D F Y K R L I N K A K [Aeea] [Aeea] [hArg] r Q[hArg] P r [NMeLeu] S H K G [Oic] [pI-Phe] P [D-Bip] {COOH} 95{Acetyl-NH} Q [NMehArg] P [NMeArg] [aMeLeu] S H K G P [Nle] P [4-Cl— F]{COOH}

In some embodiments of the invention, the modified apelin polypeptidescomprise the amino acid sequence: [z]X₁ X₂[hArg][hArg]X₃X₄X₅X₆X₇X₈X₉X₁₀G[Oic][Nle]X₁₁[4-Cl-F] (SEQ ID NO:720), wherein: z is acetyl, X₁ is oor O; X₂ is for F; X₃ is Q or [BLeu]; X₄ is [hArg] or [rhArg]; X₅ is Por [aMePro]; X₆ is [hArg] or [rhArg]; X₇ is [aMeLeu], [rCha], [BLeu], or[Cha]; X₈ is [NhSerG], [aMeS], [rSer], [DrSer], or S; X₉ is H or [rHis];X₁₀ is K, [NLysG], [rLys], or [aMeOrn]; and X₁₁ is P or [aMePro]. Inthese and other embodiments, the modified apelin polypeptide comprisesan amino acid sequence selected from SEQ ID NOs: 29-44.

The present invention also includes variants of the modified apelinpolypeptides described herein. Variants of the disclosed apelinpolypeptides may be generated by making amino acid additions orinsertions, amino acid deletions, amino acid substitutions, and/orchemical derivatives of amino acid residues within the apelinpolypeptide sequence. Desired amino acid substitutions (whetherconservative or non-conservative) can be determined by those skilled inthe art in accordance with guidance provided herein for increasingstability, while maintaining or enhancing potency of the apelinpolypeptides. In certain embodiments, conservative amino acidsubstitutions can encompass non-naturally occurring amino acid residueswhich are typically incorporated by chemical peptide synthesis ratherthan by synthesis in biological systems.

Conservative modifications can produce peptides having functional,physical, and chemical characteristics similar to those of the peptidefrom which such modifications are made. In contrast, substantialmodifications in the functional and/or chemical characteristics ofpeptides may be accomplished by selecting substitutions in the aminoacid sequence that differ significantly in their effect on maintaining(a) the structure of the molecular backbone in the region of thesubstitution, for example, as an α-helical conformation, (b) the chargeor hydrophobicity of the molecule at the target site, or (c) the size ofthe molecule. For example, a “conservative amino acid substitution” mayinvolve a substitution of a native amino acid residue with a non-nativeresidue such that there is little or no effect on the polarity or chargeof the amino acid residue at that position. Furthermore, any nativeresidue in the polypeptide may also be substituted with alanine, as hasbeen previously described for “alanine scanning mutagenesis” (see, forexample, MacLennan et al., Acta Physiol. Scand. Suppl., 643:55-67, 1998;Sasaki et al., Adv. Biophys. 35:1-24, 1998, which discuss alaninescanning mutagenesis).

Naturally occurring residues may be divided into classes based on commonside chain properties:

-   -   hydrophobic: norleucine (Nor or Nle), Met, Ala, Val, Leu, Ile;    -   neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   acidic: Asp, Glu;    -   basic: His, Lys, Arg;    -   residues that influence chain orientation: Gly, Pro; and    -   aromatic: Trp, Tyr, Phe.

Conservative amino acid substitutions may involve exchange of a memberof one of these classes with another member of the same class.Non-conservative substitutions may involve the exchange of a member ofone of these classes for a member from another class.

In making such changes, according to certain embodiments, thehydropathic index of amino acids may be considered. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics. They are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art(see, for example, Kyte et al., J. Mol. Biol. 157:105-131, 1982). It isknown that certain amino acids may be substituted for other amino acidshaving a similar hydropathic index or score and still retain a similarbiological activity. In making changes based upon the hydropathic index,in certain embodiments, the substitution of amino acids whosehydropathic indices are within ±2 is included. In certain embodiments,those that are within ±1 are included, and in certain embodiments, thosewithin ±0.5 are included

Substitution of like amino acids can be made effectively on the basis ofhydrophilicity. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, in certain embodiments, the substitution of aminoacids whose hydrophilicity values are within ±2 is included, in certainembodiments, those that are within ±1 are included, and in certainembodiments, those within ±0.5 are included.

Examples of conservative substitutions include the substitution of onenon-polar (hydrophobic) amino acid residue such as isoleucine, valine,leucine norleucine, alanine, or methionine for another non-polarresidue, the substitution of one polar (hydrophilic) amino acid residuefor another polar residue, such as between arginine and lysine, betweenglutamine and asparagine, between glycine and serine, the substitutionof one basic amino acid residue such as lysine, arginine or histidinefor another basic residue, or the substitution of one acidic residue,such as aspartic acid or glutamic acid for another acidic residue. Thephrase “conservative amino acid substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residue,provided that such polypeptide displays the requisite bioactivity. Otherexemplary amino acid substitutions that can be used in generatingvariants of the modified apelin polypeptides disclosed herein are setforth in Table 5 below.

TABLE 5 Some Useful Amino Acid Substitutions ORIGINAL RESIDUES EXEMPLARYSUBSTITUTIONS Ala Val, Leu, Ile, Gly Arg Lys, Gln, Asn, His Asn Gln AspGlu Cys Ser, Ala Gln Asn Glu Asp Gly Pro, Ala His Asn, Gln, Lys, Arg IleLeu, Val, Met, Ala, Phe, Norleucine Leu Norleucine, Ile, Val, Met, Ala,Phe Lys Arg, 1,4-Diamino-butyric Acid, Gln, Asn, His Met Leu, Phe, IlePhe Leu, Val, Ile, Ala, Tyr Pro Ala Ser Thr, Ala, Cys Thr Ser Trp Tyr,Phe Tyr Trp, Phe, Thr, Ser Val Ile, Met, Leu, Phe, Ala, Norleucine

Variants of the modified apelin polypeptides described herein can alsobe generated by derivatizing amino acids within the polypeptidesequence. The terms “derivatizing” and “derivative” or “derivatized”should be understood to mean processes and resulting compoundsrespectively in which a portion of the parent molecule has been modifiedchemically. Examples can include: (1) a compound having a cyclicportion; for example, cross-linking between residues within thecompound; (2) a compound cross-linked or having a cross-linking site;for example, the compound has a cysteinyl residue and thus formscross-linked dimers; (3) one or more peptidyl linkages are replaced by anon-peptidyl linkage; (4) an N-terminus is modified with agents capableof reacting with the amino group; (5) the C-terminus can be replacedwith an amide or ester; and (6) compounds in which individual amino acidmoieties are modified through treatment with agents capable of reactingwith selected side chains or terminal residues.

In various embodiments, the apelin polypeptide and/or vehicle/carrierportion of the compounds described herein may be derivatized. Suchderivatives may improve the solubility, absorption, stability,biological half-life, and the like of the compounds. The derivativemoieties may alternatively eliminate or attenuate any undesirableeffects of the compounds, such as induction of mast cell degranulation.Various derivitization strategies, which have been used in making themodified apelin polypeptides of the invention and are particularlyuseful for increasing the stability of apelin peptides, are set forth inFIG. 3 .

“Chemical derivative” or “chemically derivatized” also refers to asubject peptide having one or more residues chemically derivatized byreaction of a functional side group. Such derivatized molecules include,for example, those molecules in which free amino groups have beenderivatized to form amine hydrochlorides, p-toluene sulfonyl groups,carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups orformyl groups. Free carboxyl groups may be derivatized to form salts,methyl and ethyl esters or other types of esters or hydrazides. Freehydroxyl groups may be derivatized to form O-acyl or O-alkylderivatives. The imidazole nitrogen of histidine may be derivatized toform N-im-benzylhistidine. Also included as chemical derivatives arethose peptides which contain one or more naturally occurring amino acidderivatives of the twenty canonical amino acids, whether in L- orD-form. For example, 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine.

Useful derivatizations include, in some embodiments, those in which theamino terminal of the peptide is chemically blocked so that conjugationwith a vehicle or carrier will be prevented from taking place at anN-terminal free amino group. There may also be other beneficial effectsof such a modification, for example a reduction in the apelin peptideanalog's susceptibility to enzymatic proteolysis. The N-terminus can beacylated or modified to a substituted amine, or derivatized with anotherfunctional group, such as an aromatic moiety (e.g., an indole acid,benzyl (Bzl or Bn), dibenzyl (DiBzl or Bn₂), or benzyloxycarbonyl (Cbzor Z)), N,N-dimethylglycine or creatine. For example, in someembodiments, an acyl moiety, such as, but not limited to, a formyl,acetyl (Ac), propanoyl, butanyl, heptanyl, hexanoyl, octanoyl, ornonanoyl, can be covalently linked to the N-terminal end of the peptide,which can prevent undesired side reactions during conjugation of thevehicle or carrier to the peptide. Other exemplary N-terminal derivativegroups include —NRR¹ (other than —NH₂), —NRC(O)R¹, —NRC(O)OR¹,—NRS(O)₂R¹, —NHC(O)NHR¹, succinimide, or benzyloxycarbonyl-NH-(Cbz-NH—),wherein R and R¹ are each independently hydrogen or lower alkyl andwherein the phenyl ring may be substituted with 1 to 3 substituentsselected from C₁-C₄ alkyl, C₁-C₄ alkoxy, chloro, and bromo.

In some embodiments, one or more peptidyl [—C(O)NR—] linkages (bonds)between amino acid residues can be replaced by a non-peptidyl linkage.Exemplary non-peptidyl linkages are —CH₂-carbamate [—CH₂—OC(O)NR—],phosphonate, —CH₂-sulfonamide [—CH₂—S(O)₂NR—], urea [—NHC(O)NH—],—CH₂-secondary amine, and alkylated peptide [—C(O)NR⁶— wherein R⁶ islower alkyl].

In some embodiments, one or more individual amino acid residues can bederivatized. Various derivatizing agents are known to react specificallywith selected sidechains or terminal residues, as described in detailbelow by way of example.

Lysinyl residues and amino terminal residues may be reacted withsuccinic or other carboxylic acid anhydrides, which reverse the chargeof the lysinyl residues. Other suitable reagents for derivatizingalpha-amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride;trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; andtransaminase-catalyzed reaction with glyoxylate.

Arginyl residues may be modified by reaction with any one or combinationof several conventional reagents, including phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization ofarginyl residues requires that the reaction be performed in alkalineconditions because of the high pKa of the guanidine functional group.Furthermore, these reagents may react with the groups of lysine as wellas the arginine epsilon-amino group.

Specific modification of tyrosyl residues has been studied extensively,with particular interest in introducing spectral labels into tyrosylresidues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizole andtetranitromethane are used to form 0-acetyl tyrosyl species and 3-nitroderivatives, respectively.

Carboxyl sidechain groups (aspartyl or glutamyl) may be selectivelymodified by reaction with carbodiimides (R′—N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Cysteinyl residues can be replaced by amino acid residues or othermoieties either to eliminate disulfide bonding or, conversely, tostabilize cross-linking. See, e.g., Bhatnagar et al., J. Med. Chem.,39:3814-3819, 1996.

Derivatization with bifunctional agents is useful for cross-linking thepeptides or their functional derivatives to a water-insoluble supportmatrix, if desired, or to other macromolecular vehicles. Commonly usedcross-linking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimideesters, for example, esters with 4-azidosalicylic acid, homobifunctionalimidoesters, including disuccinimidyl esters such as3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimides suchas bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio] propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates, e.g., asdescribed in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642;4,229,537; and 4,330,440, are employed for protein immobilization.

Other possible modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, oxidation of the sulfur atom in Cys, methylation of thealpha-amino groups of lysine, arginine, and histidine side chains.Creighton, Proteins: Structure and Molecule Properties, W. H. Freeman &Co., San Francisco, 79-86, 1983.

The above examples of derivatizations are not intended to be anexhaustive treatment, but merely illustrative.

Recombinant DNA- and/or RNA-mediated protein expression and proteinengineering techniques, or any other methods of preparing peptides, areapplicable to the making of the apelin polypeptides disclosed herein.The term “recombinant” should be understood to mean that the material(e.g., a nucleic acid or a polypeptide) has been artificially orsynthetically (i.e., non-naturally) altered by human intervention. Thealteration can be performed on the material within, or removed from, itsnatural environment or state. For example, a “recombinant nucleic acid”is one that is made by recombining nucleic acids, e.g., during cloning,DNA shuffling or other well-known molecular biological procedures.Examples of such molecular biological procedures are found in Maniatiset al., Molecular Cloning. A Laboratory Manual. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1982. A “recombinant DNAmolecule,” is comprised of segments of DNA joined together by means ofsuch molecular biological techniques. The term “recombinant protein” or“recombinant polypeptide” as used herein refers to a protein moleculewhich is expressed using a recombinant DNA molecule. A “recombinant hostcell” is a cell that contains and/or expresses a recombinant nucleicacid.

The peptides of the invention may be made in transformed host cellsaccording to methods known to those of skill in the art. Briefly, arecombinant DNA molecule, or construct, coding for the peptide isprepared. Methods of preparing such DNA molecules are well known in theart. For instance, sequences encoding the peptides can be excised fromDNA using suitable restriction enzymes. Any of a large number ofavailable and well-known host cells may be used in the practice ofvarious embodiments. The selection of a particular host is dependentupon a number of factors recognized by the art. These include, forexample, compatibility with the chosen expression vector, toxicity ofthe peptides encoded by the DNA molecule, rate of transformation, easeof recovery of the peptides, expression characteristics, bio-safety andcosts. A balance of these factors should be struck with theunderstanding that not all hosts may be equally effective for theexpression of a particular DNA sequence. Within these generalguidelines, useful microbial host cells in culture include bacteria(such as Escherichia coli sp.), yeast (such as Saccharomyces sp.) andother fungal cells, insect cells, plant cells, mammalian (includinghuman) cells, e.g., CHO cells and HEK293 cells. Modifications can bemade at the DNA level, as well. The peptide-encoding DNA sequence may bechanged to codons more compatible with the chosen host cell. For E.coli, optimized codons are known in the art. Codons can be substitutedto eliminate restriction sites or to include silent restriction sites,which may aid in processing of the DNA in the selected host cell. Next,the transformed host is cultured and purified. Host cells may becultured under conventional fermentation conditions so that the desiredcompounds are expressed. Such fermentation conditions are well known inthe art. In addition, the DNA optionally further encodes, 5′ to thecoding region of a fusion protein, a signal peptide sequence (e.g., asecretory signal peptide) operably linked to the expressed peptideanalog. For further examples of appropriate recombinant methods andexemplary DNA constructs useful for recombinant expression of thecompositions by mammalian cells, including dimeric Fc fusion proteins(“peptibodies”) or chimeric immunoglobulin (light chain+heavy chain)-Fcheterotrimers (“hemibodies”), see, e.g., Sullivan et al., Toxin PeptideTherapeutic Agents, U.S. Patent Publication No. 2007/0071764 andSullivan et al., Toxin Peptide Therapeutic Agents, WO 2008/088422, whichare both incorporated herein by reference in their entireties.

The modified apelin polypeptides of the invention can also be made bysynthetic methods. Solid phase synthesis can be used as a technique ofmaking individual peptides since it is the most cost-effective method ofmaking small peptides. For example, well known solid phase synthesistechniques include the use of protecting groups, linkers, and solidphase supports, as well as specific protection and deprotection reactionconditions, linker cleavage conditions, use of scavengers, and otheraspects of solid phase peptide synthesis. Suitable techniques are wellknown in the art. See, e.g., Merrifield, Chem. Polypeptides, Katsoyannisand Panayotis eds., pp. 335-361, 1973; Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Davis et al., Biochem. Intl. 10:394-414, 1985; Stewart andYoung, Solid Phase Peptide Synthesis, 1969; U.S. Pat. No. 3,941,763;Finn et al., The Proteins, 3rd ed., 2:105-253, 1976; and Erickson etal., The Proteins, 3rd ed., 2: 257-527, 1976; “Protecting Groups inOrganic Synthesis,” 3rd ed., T. W. Greene and P. G. M. Wuts, Eds., JohnWiley & Sons, Inc., 1999; NovaBiochem Catalog, 2000; “SyntheticPeptides, A User's Guide,” G. A. Grant, Ed., W.H. Freeman & Company, NewYork, N.Y., 1992; “Advanced Chemtech Handbook of Combinatorial & SolidPhase Organic Chemistry,” W. D. Bennet, J. W. Christensen, L. K.Hamaker, M. L. Peterson, M. R. Rhodes, and H. H. Saneii, Eds., AdvancedChemtech, 1998; “Principles of Peptide Synthesis, 2nd ed.,” M.Bodanszky, Ed., Springer-Verlag, 1993; “The Practice of PeptideSynthesis, 2nd ed.,” M. Bodanszky and A. Bodanszky, Eds.,Springer-Verlag, 1994; “Protecting Groups,” P. J. Kocienski, Ed., GeorgThieme Verlag, Stuttgart, Germany, 1994; “Fmoc Solid Phase PeptideSynthesis, A Practical Approach,” W. C. Chan and P. D. White, Eds.,Oxford Press, 2000; G. B. Fields et al., Synthetic Peptides: A User'sGuide, 77-183, 1990. For further examples of synthetic and purificationmethods known in the art, which are applicable to making thepolypeptides and conjugates of the invention, see, e.g., Sullivan et al,U.S. Patent Publication No. 2007/0071764 and Sullivan et al., WO2008/088422 A2, which are both incorporated herein by reference in theirentireties.

The modified apelin polypeptides of the invention may be covalentlyfused, attached, linked or conjugated to one or more half-life extendingmoieties or vehicles. A “half-life extending moiety,” which is usedinterchangeably herein with “vehicle,” refers to a molecule thatprevents or mitigates in vivo degradation by proteolysis or otheractivity-diminishing chemical modification, increases in vitro or invivo half-life or other pharmacokinetic properties, such as but notlimited to decreasing the rate of renal or hepatic clearance, increasingthe rate of absorption, reducing toxicity, reducing immunogenicity,improving solubility, increasing biological activity and/or targetselectivity of the apelin peptide with respect to a target of interest,and/or increases manufacturability, compared to an unconjugated form ofthe apelin peptide. The half-life extending moiety can be one that ispharmaceutically acceptable.

A composition that includes an apelin peptide or polypeptide covalentlylinked, attached, or bound, either directly or indirectly through alinker moiety, to another peptide, vehicle, or a half-life extendingmoiety is a “conjugate” or “conjugated” molecule, whether conjugated bychemical means (e.g., post-translationally or post-synthetically) or byrecombinant fusion. Conjugation of the apelin polypeptides to thehalf-life extending moiety, or moieties, can be via the N-terminusand/or C-terminus of the apelin peptide, or can be intercalary as to thepeptide's primary amino acid sequence. In certain embodiments, thehalf-life extending moiety is conjugated to the N-terminus of themodified apelin polypeptide.

The half-life extending moiety or vehicle can be selected such that theresulting peptide conjugate achieves a sufficient hydrodynamic size toprevent clearance by renal filtration in vivo. For example, a half-lifeextending moiety can be selected that is a polymeric macromolecule,which is substantially straight chain, branched-chain (br), or dendriticin form. Alternatively, a half-life extending moiety can be selectedsuch that, in vivo, the resulting peptide conjugate will bind to a serumprotein to form a complex, such that the complex thus formed avoidssubstantial renal clearance. The half-life extending moiety or vehiclecan be, for example, a polymer (e.g., polyethylene glycol (PEG)); alipid; a cholesterol group (such as a steroid); a carbohydrate oroligosaccharide (e.g., dextran); any natural or synthetic protein (e.g.,an antibody or Fc domain), or any polypeptide or peptide that binds to asalvage receptor.

Exemplary half-life extending moieties that can be used, in accordancewith the present invention, include an immunoglobulin, an immunoglobulinFc domain, or a portion thereof, or a biologically suitable polymer orcopolymer, for example, a polyalkylene glycol compound, such as apolyethylene glycol (PEG) or a polypropylene glycol. Other appropriatepolyalkylene glycol compounds include, but are not limited to, chargedor neutral polymers of the following types: dextran, polylysine,colominic acids or other carbohydrate based polymers, polymers of aminoacids, and biotin derivatives. In some embodiments, an immunoglobulin(including light and heavy chains) or a portion thereof, can be used asa half-life-extending moiety, preferably an immunoglobulin of humanorigin, and including any of the immunoglobulins, such as, but notlimited to, IgG1, IgG2, IgG3 or IgG4.

Other examples of the half-life extending moiety or vehicle, inaccordance with the invention, include a copolymer of ethylene glycol, acopolymer of propylene glycol, a carboxymethylcellulose, a polyvinylpyrrolidone, a poly-1,3-dioxolane, a poly-1,3,6-trioxane, an ethylenemaleic anhydride copolymer, a polyaminoacid (e.g., polylysine orpolyornithine), a dextran n-vinyl pyrrolidone, a poly n-vinylpyrrolidone, a propylene glycol homopolymer, a propylene oxide polymer,an ethylene oxide polymer, a polyoxyethylated polyol, a polyvinylalcohol, a linear or branched glycosylated chain, a polyacetal, a longchain fatty acid, a long chain hydrophobic aliphatic group, or apolysialic acid (e.g., PolyXen™ technology; Gregoriadis et al.,Improving the therapeutic efficacy of peptides and proteins: a role forpolysialic acids, Intl. J. Pharmaceutics, 300:125-130, 2005,incorporated herein by reference in its entirety).

In other embodiments, the half-life extending moiety is an anionicallycharged chemical entity, covalently linked to the N-terminus of theapelin peptide. Anionically charged chemical entities include, but arenot limited to, phosphotyrosine, phosphoserine,p-phosphono(difluoro-methyl)-phenylalanine (Pfp),p-phosphono-methyl-phenylalanine (Pmp), p-phosphatidyl-phenylalanine(Ppa), or p-phosphono-methylketo-phenylalanine (Pkp), which can becovalently linked to the N-terminal of the apelin peptide, optionallyindirectly, via an AEEA linker or other linker as described herein. SeeWO 2006/042151 A2; Beeton et al., “Targeting effector memory T cellswith a selective peptide inhibitor of Kv1.3 channels for therapy ofautoimmune diseases”, Molec. Pharmacol. 67(4):1369-1381, 2005;Pennington et al., “Engineering a stable and selective peptide blockerof the Kv1.3 channel in T lymphocytes”, Molecular Pharmacology FastForward, published Jan. 2, 2009 as doi:10.1124/mol.108.052704, all ofwhich are incorporated herein by reference in their entireties.

Other embodiments of the half-life extending moiety, in accordance withthe invention, include peptide ligands or small (organic) moleculeligands that have binding affinity for a long half-life serum proteinunder physiological conditions of temperature, pH, and ionic strength.Examples include an albumin-binding peptide or small molecule ligand, atransthyretin-binding peptide or small molecule ligand, athyroxine-binding globulin-binding peptide or small molecule ligand, anantibody-binding peptide or small molecule ligand, or another peptide orsmall molecule that has an affinity for a long half-life serum protein,such as serum albumin. See, e.g., Kratz, “Albumin as a drug carrier:Design of prodrugs, drug conjugates and nanoparticles”, J. ControlledRelease, 132:171-183, 2008; Dennis et al., “Albumin binding as a generalstrategy for improving the pharmacokinetics of proteins”, J. Biol. Chem.277(38):35035-35043, 2002; Knudsen et al., “Potent derivative ofglucagon-like Peptide-1 with pharmacokinetic properties suitable foronce daily administration”, J. Med. Chem. 43:1664-1669, 2000; Kurtzhalset al., “Albumin binding of insulins acylated with fatty acids:characterization of the ligand-protein interaction and correlationbetween binding affinity and timing of the insulin effect in vivo”,Biochem. J. 312:725-731, 1995; Kenyon et al., “13C NMR Studies of thebinding of medium-chain fatty acids to human serum albumin”, J. LipidRes. 35:458-467, 1994; Blaney et al., “Method and compositions forincreasing the serum half-life of pharmacologically active agents bybinding to transthyretin-selective ligands”, U.S. Pat. No. 5,714,142;Sato et al., “Serum albumin binding moieties”, U.S. Patent PublicationNo. 2003/0069395 A1; and U.S. Pat. No. 6,342,225. Exemplary smallmolecule ligands that can bind to human serum albumin include, but arenot limited to, pIFBu ((S)-2-(4-(4-iodophenyl)butanamido)pentanedioicacid) and DOTA(2,2′,2″,2′″-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraaceticacid). Such ligands can be attached to the modified apelin polypeptidesof the invention, such as in polypeptide of SEQ ID NO: 118({pIFBu}[AC4Abu][Aeea][Aeea]QRP[NMeArg]LSHKGP[Nle]P[4-Cl-F]{COOH}), toincrease serum half-life.

A “long half-life serum protein” is one of the hundreds of differentproteins dissolved in mammalian blood plasma, including so-called“carrier proteins” (such as albumin, transferrin and haptoglobin),fibrinogen and other blood coagulation factors, complement components,immunoglobulins, enzyme inhibitors, precursors of substances such asangiotensin and bradykinin and many other types of proteins. Theinvention encompasses the use of any single species of pharmaceuticallyacceptable half-life extending moiety, such as, but not limited to,those described herein, or the use of a combination of two or moredifferent half-life extending moieties, such as PEG and immunoglobulinFc domain or a portion thereof (see, e.g., Feige et al., “Modifiedpeptides as therapeutic agents”, U.S. Pat. No. 6,660,843), such as a CH2domain of Fc, albumin (e.g., human serum albumin (HSA); see, e.g.,Ehrlich et al., “Preparation and characterization of albumin conjugatesof a truncated Peptide YYY analogue for half-life extension”,Bioconjugate Chem., 24(12):2015-2024, 2013; U.S. Pat. No. 6,926,898;U.S. Patent Publication No. 2005/0054051; U.S. Pat. No. 6,887,470), atransthyretin (TTR; see, e.g., U.S. Patent Publication Nos. 2003/0195154A1 and 2003/0191056 A1), or a thyroxine-binding globulin (TBG), or acombination such as immunoglobulin(light chain+heavy chain) and Fcdomain (the heterotrimeric combination a so-called “hemibody”), forexample as described in WO 2008/088422, which is incorporated herein byreference in its entirety.

Extending the half-life of the modified apelin polypeptide can also bereferred to as increased “stability.” Increased stability can also beunderstood to mean reduced clearance of the apelin polypeptide oroverall increased exposure of the apelin polypeptide or an apelinpolypeptide with a modification or combination of modifications thatreduces the rate of metabolic degradation relative to unmodified apelinpolypeptide. Increased stability can prolong half-life of the apelinpolypeptide in biological matrices such as plasma and tissue homogenatesin vitro and prolong plasma residence times in vivo. In vivo plasmaresidence times should be understood to mean circulating life-time ofthe intact peptide drug entity administered into an animal. In vitroplasma residence times should be understood to mean life-time of theintact peptide drug entity after addition in biological medium.

Modifications of the apelin polypeptide can also lead to increased“potency” of the molecule, e.g. improving the pharmacokinetic orpharmacodynamic properties of the molecule. Increased potency can alsobe understood to mean an apelin polypeptide with a modification orcombination of modifications that reduces the rate of metabolicdegradation relative to unmodified apelin polypeptide. Increasedstability can prolong half-life of the apelin polypeptide in biologicalmatrices such as plasma and tissue homogenates in vitro and prolongplasma residence times in vivo. Increased potency may further beunderstood to mean increased affinity and/or efficacy of the modifiedapelin polypeptide for the APJ receptor.

In certain embodiments, one or more modified apelin polypeptidesdescribed herein is conjugated or attached to a half-life extensionmoiety or vehicle through the polypeptide's N-terminus, C-terminus,backbone, or side-chain via chemical modification in variousconfigurations. Thus, in one embodiment, the vehicle-peptide conjugatesmay be described by the following formula I:

wherein:V¹ is a vehicle (e.g. a PEG, lipid, or other half-life extensionmoiety);

-   -   A¹, A², A³, and A⁴ are each independently selected from        -(L¹)_(e)-P¹, -(L¹)_(e)-P¹-(L²)_(f)-P₂,        -(L¹)_(e)-P¹-(L²)_(f)-P²-(L³)_(g)-P³,        -(L¹)_(e)-P¹-(L²)_(f)-P²-(L³)_(g)-P³-(L⁴)_(h)-P⁴ and higher        multimers thereof;

P¹, P², P³, and P⁴ are each independently sequences of apelinpolypeptides and can be any of the apelin peptides described in thisapplication without the lipid and/or Aeea and/or γ-glutamate and/oranother moiety that comprises a portion of the conjugation linker, e.g.the apelin peptides with amino acid sequences of SEQ ID NOs: 121-647;

-   -   L¹, L², L³, and L⁴ are each independently linkers; and    -   a, b, c, d, e, f, g, and h are each independently 0 or 1,        provided that at least one of a, b, and c is 1.

In another embodiment, the vehicle-peptide conjugate is described by thefollowing formula II:V¹-A¹and multimers thereof wherein V¹ is a PEG, lipid, or other half-lifeextension moiety, and is attached, with or without a linker, at theN-terminus of A²;

In another embodiment, the vehicle-peptide conjugate is described by thefollowing formula III:

and multimers thereof wherein V¹ is a PEG, lipid, or other half-lifeextension moiety, and is attached, with or without a linker, at thebackbone or side-chain of A²;

In another embodiment, the vehicle-peptide conjugate is described by thefollowing formula IV:A³-V¹and multimers thereof wherein V¹ is a PEG, lipid, or other half-lifeextension moiety, and is attached, with or without a linker, at theC-terminus of A³;

In another embodiment, the vehicle-peptide conjugate is described by thefollowing formula V:A²-V¹-A¹and multimers thereof wherein V¹ is a PEG, lipid, or other half-lifeextension moiety, and is attached, with or without a linker, at anylocation of A¹ and A²;

In another embodiment, the vehicle-peptide conjugate is described by thefollowing formula VI;

and multimers thereof wherein V¹ is a PEG, lipid, or other half-lifeextension moiety, and is attached, with or without a linker, at anylocation of A¹, A², A³, or A⁴;

In another embodiment, the vehicle-peptide conjugate is described by thefollowing formula VII:

and multimers thereof wherein V¹ and V² are a PEG, lipid, or otherhalf-life extension moiety, and are attached, with or without a linker,at any location of A¹.

In certain embodiments, γ-glutamate may be inserted into a modifiedapelin polypeptide of the invention. The γ-glutamate may be insertedbetween a lipid moiety (e.g. a fatty acid) and the modified apelinpolypeptide. In addition to γ-glutamate, any other moiety may be presentas a constituent of the conjugation linker as described in furtherdetail herein. A constituent of the conjugation linker may be a polar,non-polar, hydrophobic, aliphatic, or aromatic moiety that serves aspecial, functional, and/or structural role.

In certain embodiments, the half-life extension moiety or vehicle is alipid. Thus, any of the modified apelin polypeptides disclosed hereincan be conjugated, optionally through a conjugation linker, to a lipidmoiety, such as a fatty acid. In some embodiments, the fatty acid is aC₁ to C₂₅ saturated or unsaturated fatty acid. Exemplary fatty acidsthat can be used as half-life extension moieties and conjugated to themodified apelin polypeptides of the invention, optionally through aconjugation linker, are listed in Table 6 below.

TABLE 6 Exemplary Fatty Acid Moieties COMMON CHEMICAL CHEMICAL NAME NAMESTRUCTURE ABBREVIATION Butyric acid Butanoic acid CH₃(CH₂)₂COOH ButanoylCaproic acid Hexanoic acid CH₃(CH₂)₄COOH Hexanoyl Caprylic acid Octanoicacid CH3(CH2)₆COOH Oc Capric acid decanoic acid CH₃(CH₂)₈COOH DecanoylLaurie acid dodecanoic acid CH₃(CH₂)₁₀COOH Dodecanoyl, Dodc Tridecylicacid Tridecanoic acid CH₃(CH₂)₁₁COOH TDA Myristoic acid tetradecanoicacid CH₃(CH₂)₁₂COOH Myristyl, Myrist Pentadecylic acid Pentadecanoicacid CH₃(CH₂)₁₃COOH PDA Palmitic acid Hexadecanoic acid CH₃(CH₂)₁₄COOHPalm margaric acid Heptadecanoic acid CH₃(CH₂)₁₅COOH HDA Stearic acidOctadecanoic acid CH₃(CH₂)₁₆COOH St Arachidic acid icosanoic acidCH₃(CH₂)₁₈COOH Behenic acid docosanoic acid CH₃(CH₂)₂₀COOH Lignocericacid tetracosanoic acid CH₃(CH₂)₂₂COOH Cerotic acid hexacosanoic acidCH₃(CH₂)₂₄COOH Succinic acid butanedioic acid HOOC(CH₂)₂COOHSuccinicacid adipic acid hexanedioic acid HOOC(CH₂)₄COOH AdpA Subericacid octanedioic acid HOOC(CH₂)₆COOH Subericacid Sebacic aciddecanedioic acid HOOC(CH₂)₈COOH Sebacicacid DDDA dodecanedioic acidHOOC(CH₂)₁₀COOH DDDA tetradecanedioic acid HOOC(CH₂)₁₂COOH TetDDAhexadecanedioic acid HOOC(CH₂)₁₄COOH HexDDA Octadecandioic acidHOOC(CH₂)₁₆COOH ODDA

In some embodiments, the fatty acid or fatty acyl group conjugated to amodified apelin polypeptide of the invention is selected from Butanoyl,Hexanoyl, Octanoyl, Decanoyl, Dodecanoyl, Tridecanoyl, Tetradecanoyl,Pentadecanoyl, Hexadecanoyl, Heptadecanoyl, Octadecanoyl,Octadecandioyl, Octanedioyl, Decanedioyl, Dodecanedioyl, Hexanedioyl,Butanedioyl, Tetradecanedioyl, or Hexadecanedioyl. In other embodiments,the fatty acid or fatty acyl group conjugated to a modified apelinpolypeptide of the invention is selected from Octadecandioyl,Heptadecanoyl, Tridecanoyl, Butanoyl, Hexanoyl, Hexadecanoyl,Butanedioyl, Octanedioyl, or Decanedioyl. In certain embodiments, thefatty acid or fatty acyl group conjugated to a modified apelinpolypeptide of the invention is selected from Tridecanoyl, Butanoyl,Hexanoyl, Hexadecanoyl, Butanedioyl, Octanedioyl, or Decanedioyl. Incertain other embodiments, the fatty acid or fatty acyl group conjugatedto a modified apelin polypeptide of the invention is selected fromOctanoyl, Decanoyl, Dodecanoyl, Tridecanoyl, Tetradecanoyl,Pentadecanoyl, Hexadecanoyl, Heptadecanoyl, Octadecanoyl, orOctadecandioyl.

In one embodiment of the invention, the lipid-apelin peptide conjugatecan be described by the following formula:X₁X₂X₃X₄  (SEQ ID NO:721)wherein:

X₁ is a fatty acyl group,

X₂ is γGlu or is absent,

X₃ is a PEG group or is absent, and

X₄ is an apelin polypeptide.

The amino acid residues in the apelin peptide of the above formula canbe L- or D-amino acids, α- or β-amino acids, non-canonical amino acidsor the L- or D- or α- or β-forms of the non-canonical amino acids. Theapelin polypeptide in the above formula can be any of the modifiedapelin polypeptides described herein. In one embodiment, the PEG groupis one or more AEEA ([2-(2-Amino-ethoxy)-ethoxy]-acetic acid) groups. Inrelated embodiments, X₂ is γGlu (also referred to herein as AC4Abu) andX₃ is AEEA or AEEA-AEEA.

In another embodiment of the invention, the lipid-apelin peptideconjugate can be described by the following formula:{HDA}[AC4Abu][Aeea][Aeea]X₁X₂X₃P[Cha]SHKG[Oic][Nle]X₄ X₅  (SEQ ID No.722),wherein: X₁ is [hArg] or is absent, X₂ is Q or [q], X₃ is [hArg] or [r],X₄ is P or [Pip] and X₅ is [4-Cl-F] or [D-4ClF]. In these and otherembodiments, the lipid-peptide conjugate may comprise the sequence ofSEQ ID NO: 237, 239, or 242.

In yet another embodiment of the invention, the lipid-apelin peptideconjugate can be described by the following formula:{HDA}[AC4Abu][Aeea][Aeea]X₁X₂X₃PX₄X₅SHKG[Oic][Nle]X₆X₇  (SEQ ID No. 723)wherein: X₁ is [hArg] or is absent, X₂ is Q or [q], X₃ is r or [hArg],X₄ is r or [NMeArg], X₅ is [NMeLeu] or [Cha], X₆ is [Pip] or P, and X₇is [4-CL-F], [f], [D-4ClF], or [Tic]. In these and other embodiments,the lipid-peptide conjugate may comprise the sequence of any one of SEQID NOs: 234-243.

In some embodiments of the invention, the lipid-apelin peptide conjugatecan be described by the following formula:{TDA}[AC4Abu][Aeea][Aeea]X₁X₂X₃X₄ X₅X₆ X₇X₈KG[Oic]X₉X₁₀ X₁₁  (SEQ ID NO:724)wherein: X₁ is [hArg], [r] or is absent, X₂ is Q, [q], [BLeu] or[NMeGln], X₃ is [hArg] or [r], X₄ is P, [Pip], [Oic] or [Sar], X₅ is[NMeArg], [r] or [hArg], X₆ is [Cha], [NMeLeu], [BLeu] or [NMeCha], X₇is S, [BhSer], [bAla], [NhSerG] or [aMeS], X₈ is H, A, Y, [Tle], [Deg],L or V, X₉ is [Nle] or [pl-Phe], X₁₀ is P, a D-amino acid, a β-aminoacid, a non-canonical amino acid or the D- or β-form of thenon-canonical amino acid, and X₁₁ is [4-Cl-F], [D-4ClF], [D-Bip] or isabsent. In certain embodiments, X₁₀ is [D-Tic], [4-Cl-F], [D-4ClF],[Aic], [Oic], [D-4F], [D-Ogl], [f], [1-Nal], [2-Nal], [D-Bip], [Tic],[Aib] or [Deg]. In these and other embodiments, the lipid-peptideconjugate may comprise the sequence of any one of SEQ ID NOs: 121-210and 245-256.

In another embodiment of the invention, the lipid-apelin peptideconjugate can be described by the following formula:{TDA}[AC4Abu][Aeea][Aeea]X₁q[hArg]P[NMeArg][Cha]SHKG[Oic][Nle]X₂X₃  (SEQID No: 725),wherein: X₁ is [hArg] or is absent, X₂ is P, a D-amino acid or anon-canonical amino acid, and X₃ is a D-amino acid, a non-canonicalamino acid or —COOH. In these and other embodiments, the lipid-peptideconjugate may comprise the sequence of any one of SEQ ID NOs: 121-152.

In still another embodiment of the invention, the lipid-apelin peptideconjugate can be described by the following formula:{TDA}[AC4Abu][Aeea][Aeea][hArg]q[hArg]P[NMeArg][Cha]SHKG[Oic][Nle]X₁COOH  (SEQID No. 726),wherein X₁ is a D-amino acid, a β-amino acid, a non-canonical amino acidor the D- or β-form of the non-canonical amino acid. In someembodiments, X₁ is [D-Tic], [4-CL-F], [D-4ClF], [Aic], [Oic], [D-4lF],[D-IgL], [f], [1-Nal], [2-Nal], [D-Bip] or [Tic]. In certainembodiments, the lipid-peptide conjugate may comprise the sequence ofany one of SEQ ID NOs: 166, 171, and 245-256.

In one embodiment of the invention, the lipid-apelin peptide conjugatecan be described by the following formula:{TDA}[AC4Abu][Aeea][Aeea]QX₁PX₂X₃SHKG[Oic]X₄X₅X₆  (SEQ ID No: 727),wherein: X₁ is R, r or [hArg], X₂ is r, [hArg] or [NMeArg], X₃ is[NMeLeu] or [Cha], X₄ is [pl-Phe] or [Nle], X₅ is P or [D-1Nal], and X₆is [D-Bip], [4-CL-F], [D-4ClF] or is absent. In these and otherembodiments, the lipid-peptide conjugate may comprise the sequence ofany one of SEQ ID NOs: 165 and 211-233.

In yet another embodiment of the invention, the lipid-apelin peptideconjugate can be described by the following formula:{TDA}[AC4Abu][Aeea][Aeea]X₁X₂X₃PX₄X₅X₆X₇KG[Oic]X₈X₉X₁₀  (SEQ ID No: 728)wherein: X₁ is [hArg] or [r], X₂ is Q or [q], X₃ is r or [hArg], X₄ is ror [NMeArg], X₅ is [NMeLeu], [BLeu], or [Cha], X₆ is S or [bAla], X₇ isH, A or [Tle], X₈ is [Nle] or [pl-Phe], X₉ is P, a D-amino acid, aβ-amino acid, a non-canonical amino acid or the D- or β-form of thenon-canonical amino acid, and X₁₀ is [Oic], [4-Cl-F], [D-4ClF],[D-1Nal], [D-Bip] or is absent. In some embodiments, X₉ is [D-Tic],[4-Cl-F][D-4ClF], [Aic], [Oic], [D-igl], [f], [D-1Nal], [D-2Nal],[1-Nal], [2-Nal] or [D-Bip] and X₁₀ is absent. In certain embodiments,the lipid-peptide conjugate may comprise the sequence of any one of SEQID NOs: 153-155, 166, 171, 173-175, 180, 199, 201, 208, and 245-256.

Exemplary lipid-apelin peptide conjugates of the invention are providedin Table 7 below. The present invention includes an isolated polypeptidecomprising the sequence of any one of the lipid-peptide conjugateslisted in Table 7.

TABLE 7 Exemplary Lipid-Apelin Peptide Conjugates SEQ ID NO: SEQUENCE121 {TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[Oic] 122{TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P [Aic]123 {TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[D-1Nal] 124{TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P [D-2Nal]125 {TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[D-Igl] 126{TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[f]{COOH} 127{TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P [D-4ClF]128 {TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[D-Tic] 129{TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P [D-4IF]130 {TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][D-Tic]{COOH} 131{TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][4-Cl—F]{COOH} 132{TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][D-4ClF]{COOH} 133{TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][Aic]{COOH} 134{TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][Oic]{COOH} 135{TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][D-4IF]{COOH} 136{TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][D-Igl]{COOH} 137{TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][f] {COOH}138 {TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][1-Nal]{COOH} 139{TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][2-Nal]{COOH} 140{TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][D-1Nal]{COOH} 141{TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][D-2Nal]{COOH} 142{TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][D-Bip]{COOH} 143{TDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][Tic]{COOH} 144{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[Oic] 145{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[Aic] 146{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[D-1Nal] 147{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[D-2Nal] 148{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[D-Igl] 149{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[f] 150{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[D-4ClF] 151{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[D-Tic] 152{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[D-4IF] 153{TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg]P[r][NMeLeu]SHKG[Oic][pI-Phe]P[Oic]{COOH} 154{TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg]P[r][NMeLeu]SHKG[Oic][pI-Phe]P[D-4ClF]{COOH} 155{TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg]P[r][NMeLeu]SHKG[Oic][pI-Phe]P[D-1Nal]{COOH} 156{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[r][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip] 157{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][Aib][4-Cl—F] 158{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][Deg][4-Cl—F] 159{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][Pip][4-Cl—F] 160{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][Pip][D-4ClF] 161{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg][Pip][NMeArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] 162{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg][Pip][NMeArg][Cha]SHKG[Oic][Nle][Pip][4-Cl—F] 163{TDA}[AC4Abu][Aeea][Aeea][hArg]Q[hArg]P[hArg][Cha]SHKG[Oic][Nle][D-1Nal] 164{TDA}[AC4Abu][Aeea][Aeea][hArg]Q[hArg]P[NMeArg][Cha]SHKG[Oic][Nle][D-1Nal] 165{TDA}[AC4Abu][Aeea][Aeea][hArg]Q[r]P[NMeArg][Cha]SHKG[Oic][Nle] [D-1Nal]{COOH} 166{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][D-1Nal]{COOH} 167{TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg]P[NMeArg][Cha]SHKG[Oic][Nle] [D-1Nal]168 {TDA}[AC4Abu][Aeea][Aeea][hArg]Q[hArg]P[hArg][Cha]SHKG[Oic][Nle][4-Cl—F] 169{TDA}[AC4Abu][Aeea][Aeea][hArg]Q[hArg]P[NMeArg][Cha]SHKG[Oic][Nle][4-Cl—F] 170{TDA}[AC4Abu][Aeea][Aeea][hArg]Q[r]P[NMeArg][Cha]SHKG[Oic][Nle] [4-Cl—F]171 {TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][4-Cl—F]{COOH} 172{TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg]P[NMeArg][Cha]SHKG[Oic][Nle] [4-Cl—F]173 {TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg]P[r][NMeLeu]SHKG[Oic][pI-Phe][D-Bip] 174{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[r][NMeLeu]SHKG[Oic][pI-Phe][D-Bip] {COOH} 175{TDA}[AC4Abu][Aeea][Aeea][hArg]Q[r]P[r][NMeLeu]SHKG[Oic][pI-Phe][D-Bip]{COOH} 176{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[r][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip] 177{TDA}[AC4Abu][Aeea][Aeea][hArg]Q[r]P[r][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip] 178{TDA}[AC4Abu][Aeea][Aeea][hArg][q][r]P[r][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip] 179{TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg]P[r][BLeu]SHKG[Oic][pI-Phe]P [D-Bip]180 {TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[r][BLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 181{TDA}[AC4Abu][Aeea][Aeea][hArg][q][r]P[r][BLeu]SHKG[Oic][pI-Phe]P[D-Bip] 182{TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg]P[r][NMeCha]SHKG[Oic][pI-Phe]P[D-Bip] 183{TDA}[AC4Abu][Aeea][Aeea][r][BLeu][hArg]P[r][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip] 184{TDA}[AC4Abu][Aeea][Aeea][hArg][BLeu][r]P[r][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip] 185{TDA}[AC4Abu][Aeea][Aeea][r][BLeu][r]P[r][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip] 186{TDA}[AC4Abu][Aeea][Aeea][r][NMeGln][hArg]P[r][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip] 187{TDA}[AC4Abu][Aeea][Aeea][r][NMeGln][r]P[r][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip] 188{TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg]P[r][NMeLeu]SAKG[Oic][pI-Phe]P[D-Bip] 189{TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg]P[r][NMeLeu]SYKG[Oic][pI-Phe]P[D-Bip] 190{TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg]P[r][NMeLeu]S[Tle]KG[Oic][pI-Phe]P[D-Bip] 191{TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg]P[r][NMeLeu]SLKG[Oic][pI-Phe]P[D-Bip] 192{TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg]P[r][NMeLeu]SVKG[Oic][pI-Phe]P[D-Bip] 193{TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg][Oic][r][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip] 194{TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg][Sar][r][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip] 195{TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg]P[r][NMeLeu][BhSer]HKG[Oic][pI-Phe]P[D-Bip] 196{TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg]P[r][NMeLeu][bAla]HKG[Oic][pI-Phe]P[D-Bip] 197{TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg]P[r][NMeLeu][NhSerG]HKG[Oic][pI-Phe]P[D-Bip] 198{TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg]P[r][NMeLeu][aMeS]HKG[Oic][pI-Phe]P[D-Bip] 199{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[r][NMeLeu]SAKG[Oic][pI-Phe]P[D-Bip]{COOH} 200{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[r][NMeLeu]SYKG[Oic][pI-Phe]P[D-Bip] 201{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[r][NMeLeu]S[Tle]KG[Oic][pI-Phe]P[D-Bip]{COOH} 202{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[r][NMeLeu]S[Deg]KG[Oic][pI-Phe]P[D-Bip] 203{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[r][NMeLeu]SLKG[Oic][pI-Phe]P[D-Bip] 204{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[r][NMeLeu]SVKG[Oic][pI-Phe]P[D-Bip] 205{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg][Oic][r][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip] 206{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg][Sar][r][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip] 207{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[r][NMeLeu][BhSer]HKG[Oic][pI-Phe]P[D-Bip] 208{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[r][NMeLeu][bAla]HKG[Oic][pI-Phe]P[D-Bip]{COOH} 209{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[r][NMeLeu][NhSerG]HKG[Oic][pI-Phe]P[D-Bip] 210{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[r][NMeLeu][aMeS]HKG[Oic][pI-Phe]P[D-Bip] 211{TDA}[AC4Abu][Aeea][Aeea]RQRP[r][NMeLeu]SHKG[Oic][pI-Phe]P[D- Bip] 212{TDA}[AC4Abu][Aeea][Aeea]RQRP[hArg][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 213{TDA}[AC4Abu][Aeea][Aeea][r]QRP[hArg][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 214{TDA}[AC4Abu][Aeea][Aeea]RQRP[hArg][Cha]SHKG[Oic][pI-Phe]P[D- Bip] 215{TDA}[AC4Abu][Aeea][Aeea][r]QRP [r][Cha]SHKG[Oic][pI-Phe]P[D-Bip] 216{TDA}[AC4Abu][Aeea][Aeea]RQRP[r][Cha]SHKG[Oic][pI-Phe]P[D-Bip] 217{TDA}[AC4Abu][Aeea][Aeea][r]QRP[hArg][Cha]SHKG[Oic][pI-Phe]P[D- Bip] 218{TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg]P[r][NMeLeu]SHKG[Oic][Nle]P[4-Cl—F]{COOH} 219{TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg]P[r][NMeLeu]SHKG[Oic][pI-Phe]P[4-Cl—F] 220{TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg]P[r][NMeLeu]SHKG[Oic][Nle]P [D-Bip]221 {TDA}[AC4Abu][Aeea][Aeea][r]Q[hArg]P[r][Cha]SHKG[Oic][Nle]P[4-Cl—F]{COOH} 222{TDA}[AC4Abu][Aeea][Aeea]QRP[hArg][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 223{TDA}[AC4Abu][Aeea][Aeea]QRP[hArg][Cha]SHKG[Oic][pI-Phe]P[D- Bip] 224{TDA}[AC4Abu][Aeea][Aeea]QRP[r][Cha]SHKG[Oic][pI-Phe]P[D-Bip] 225{TDA}[AC4Abu][Aeea][Aeea]Q[hArg]P[r][NMeLeu]SHKG[Oic][Nle]P[4- Cl—F] 226{TDA}[AC4Abu][Aeea][Aeea]Q[hArg]P[r][NMeLeu]SHKG[Oic][pI-Phe] P[4-Cl—F]227 {TDA}[AC4Abu][Aeea][Aeea]Q[hArg]P[r][NMeLeu]SHKG[Oic][Nle]P[D- Bip]228 {TDA}[AC4Abu][Aeea][Aeea]Q[hArg]P[r][Cha]SHKG[Oic][Nle]P[4-Cl—F]{COOH} 229{TDA}[AC4Abu][Aeea][Aeea][hArg]Q[r]P[NMeArg][Cha]SHKG[Oic][Nle]P[4-Cl—F]{COOH} 230{TDA}[AC4Abu][Aeea][Aeea][hArg]Q[r]P[NMeArg][Cha]SHKG[Oic][Nle]P[D-4ClF]{COOH} 231{TDA}[AC4Abu][Aeea][Aeea][Aeea]Q[r]P[NMeArg][Cha]SHKG[Oic][Nle]P[4-Cl—F]{COOH} 232{TDA}[AC4Abu][Aeea][Aeea][Aeea]Q[r]P[NMeArg][Cha]SHKG[Oic][Nle]P[D-4ClF]{COOH} 233{TDA}[AC4Abu][Aeea][Aeea][Aeea]Q[r]P[NMeArg][Cha]SHKG[Oic][Nle][D-1Nal]{COOH} 234 {HDA}[AC4Abu][Aeea][Aeea]Q[r]P[NMeArg][Cha]SHKG[Oic][Nle][Pip] [4-Cl—F]{COOH} 235{HDA}[AC4Abu][Aeea][Aeea][q][hArg]P[r][NMeLeu]SHKG[Oic][Nle]P[f] {COOH}236 {HDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[4-Cl—F]{COOH} 237{HDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[D-4ClF]{COOH} 238{HDA}[AC4Abu][Aeea][Aeea]Q[r]P[NMeArg][Cha]SHKG[Oic][Nle]P[Tic] {COOH}239 {HDA}[AC4Abu][Aeea][Aeea][hArg]Q[r]P[NMeArg][Cha]SHKG[Oic][Nle][Pip][4-Cl—F] {COOH} 240{HDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[r][NMeLeu]SHKG[Oic][Nle]P[f]{COOH} 241{HDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] {COOH} 242{HDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[D-4ClF] {COOH} 243{HDA}[AC4Abu][Aeea][Aeea][hArg]Q[r]P[NMeArg][Cha]SHKG[Oic][Nle]P[Tic]{COOH} 244{TDA}[AC4Abu][Aeea][Aeea]Q[r]P[NMeArg][Cha]SHKG[Oic][Nle]P[Tic] {COOH}245 {TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][D-Tic]{COOH} 246{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][D-4ClF] {COOH} 247{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][Aic]{COOH} 248{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][Oic]{COOH} 249{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][D-4IF]{COOH} 250{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][D-Igl]{COOH} 251{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][f]{COOH} 252{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][1-Nal]{COOH} 253{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][2-Nal]{COOH} 254{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][D-2Nal] {COOH} 255{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][D-Bip] {COOH} 256{TDA}[AC4Abu][Aeea][Aeea][hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle][Tic]{COOH} 257{TDA}[AC4Abu][Aeea][Aeea]OF[hArg][hArg]QRP[hArg][NMeLeu]SHKG[Oic][Nle][2Nal]{COOH} 258{PDA}[AC4Abu][Aeea][Aeea]OF[hArg][hArg]QRP[hArg][NMeLeu]SHKG[Oic][Nle][2Nal]{COOH} 259{HDA}[AC4Abu][Aeea][Aeea]OF[hArg][hArg]QRP[hArg][NMeLeu]SHKG[Oic][Nle][2Nal]{COOH} 260 {ODDA}[AC4Abu][Aeea][Aeea]OF[hArg][hArg]QRP[hArg][NMeLeu] SHKG[Oic][Nle][2Nal]{COOH} 261{TDA}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 262{PDA}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 263{HDA}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 264{ODDA}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 265{TDA}[AC4Abu][Aeea][Aeea]q[hArg]Pr[Cha]SHKG[Oic][Nle]P[D-4ClF] {COOH}266 {TDA}[AC4Abu][Aeea][Aeea]QrPr[Cha]SHKG[Oic][Nle]P[D-4ClF] {COOH} 267{TDA}[AC4Abu][Aeea][Aeea]QrP[NMeArg][Cha]SHKG[Oic][Nle]P[4-Cl— F]{COOH}268 {TDA}[AC4Abu][Aeea][Aeea]q[hArg]Pr[Cha]SHKG[Oic][Nle]P[D-1Nal]{COOH} 269 {TDA}[AC4Abu][Aeea][Aeea]q[hArg]Pr[Cha]SHKG[Oic][Nle]P[D-Tic]{COOH} 270{TDA}[AC4Abu][Aeea][Aeea]q[hArg]Pr[Cha]SHKG[Oic][Nle][Aib][4-Cl—F]{COOH} 271{TDA}[AC4Abu][Aeea][Aeea]q[hArg]Pr[Cha]SHKG[Oic][Nle][Deg][4-Cl—F]{COOH} 272{TDA}[AC4Abu][Aeea][Aeea]q[hArg]Pr[Cha]SHKG[Oic][Tle]P[4-Cl—F] {COOH}273 {TDA}[AC4Abu][Aeea][Aeea]q[hArg]Pr[Cha]S[Aib]KG[Oic][Nle]P[4-Cl—F]{COOH} 274{TDA}[AC4Abu][Aeea][Aeea]q[hArg]Pr[BLeu]SHKG[Oic][Nle]P[4-Cl—F] {COOH}275 {TDA}[AC4Abu][Aeea][Aeea]q[hArg]P[NMeArg][BLeu]SHKG[Oic][Nle]P[4-Cl—F]{COOH} 276{TDA}[AC4Abu][Aeea][Aeea]q[hArg]Pr[NMeCha]SHKG[Oic][Nle]P[4-Cl— F]{COOH}277 {TDA}[AC4Abu][Aeea][Aeea]l[hArg]Pr[Cha]SHKG[Oic][Nle]P[4-Cl—F]{COOH} 278{TDA}[AC4Abu][Aeea][Aeea][BLeu][hArg]Pr[Cha]SHKG[Oic][Nle]P[4-Cl—F]{COOH} 279{TDA}[AC4Abu][Aeea][Aeea]Q[NMeArg]Pr[Cha]SHKG[Oic][Nle]P[4-Cl— F]{COOH}280 {TDA}[AC4Abu][Aeea][Aeea]q[NMeArg]Pr[Cha]SHKG[Oic][Nle]P[4-Cl—F]{COOH} 281{TDA}[AC4Abu][Aeea][Aeea]q[hArg]Pr[BLeu]SHKG[Oic][Nle]P[D-4ClF] {COOH}282 {TDA}[AC4Abu][Aeea][Aeea]q[hArg]Pr[Cha]SHKG[Oic][Nle][4-Cl—F] {COOH}283 {TDA}[AC4Abu][Aeea][Aeea]q[hArg]Pr[Cha]SHKG[Oic][Nle][D-4ClF] {COOH}284 {TDA}[AC4Abu][Aeea][Aeea]QrP[NMeArg][Cha]SHKG[Oic][Nle]P[D-4ClF]{COOH} 285{TDA}[AC4Abu][Aeea][Aeea]QrP[NMeArg][Cha]SHKG[Oic][Nle][D- 1Nal]{COOH}286 {hexanoyl}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 287{TDA}[AC4Abu][Aeea][Aeea]QRP[NMeArg]LSHKGP[Nle]P[4-Cl—F] {COOH} 288{Sebacicacid}[AC4Abu][Aeea][Aeea]Q[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 289{Sebacicacid}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 290{ODDA}[AC4Abu][Aeea][Aeea]QRPRLSHKGPMPF{COOH} 291{Sebacicacid}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 292{TDA}[AC4Abu][Aeea][Aeea]l[hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[D-4ClF]{COOH} 293{TDA}[AC4Abu][Aeea][Aeea]q[hArg]P[NMeArg][Cha]SH[bAla]G[Oic][Nle]P[D-4ClF]{COOH} 294{TDA}[AC4Abu][Aeea][Aeea]q[hArg]P[NMeArg][Cha]SHK[bAla][Oic][Nle]P[D-4ClF]{COOH} 295{TDA}[AC4Abu][Aeea][Aeea]q[hArg]P[NMeArg][Cha]SHKG[Oic][Nle][bAla][D-4ClF]{COOH} 296{TDA}[AC4Abu][Aeea][Aeea]q[hArg]P[NMeArg][Cha]SHKG[Oic][Nle][BhPro][D-4ClF]{COOH} 297{TDA}[AC4Abu][Aeea][Aeea]OF[hArg][hArg]Q[NMeArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[4-Cl—F]{COOH} 298{TDA}[AC4Abu][Aeea][Aeea][BLeu][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[D-4ClF]{COOH} 299{TDA}[AC4Abu][Aeea][Aeea][BhAsn][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[D-4ClF]{COOH} 300{TDA}[AC4Abu][Aeea][Aeea][BhLeu][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[D-4ClF]{COOH} 301{TDA}[AC4Abu][Aeea][Aeea]q[hArg][BhPro][NMeArg][Cha]SHKG[Oic][Nle]P[D-4ClF]{COOH} 302{TDA}[AC4Abu][Aeea][Aeea]q[hArg]P[NMeArg][BhLeu]SHKG[Oic][Nle]P[D-4ClF]{COOH} 303{TDA}[AC4Abu][Aeea][Aeea]q[hArg]P[NMeArg][Cha][bAla]HKG[Oic][Nle]P[D-4ClF]{COOH} 304{TDA}[AC4Abu][Aeea][Aeea]q[hArg]P[NMeArg][Cha]S[bAla]KG[Oic][Nle]P[D-4ClF]{COOH} 305{TDA}[AC4Abu][Aeea][Aeea]q[hArg]P[NMeArg][Cha]SH[3Pal]G[Oic][Nle]P[D-4ClF]{COOH} 306{TDA}[AC4Abu][Aeea][Aeea]q[hArg]P[NMeArg][Cha]SH[BLys]G[Oic][Nle]P[D-4ClF]{COOH} 307{TDA}[AC4Abu][Aeea][Aeea]OF[hArg][hArg][BLeu][hArg]P[hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F]{COOH} 308{TDA}[AC4Abu][Aeea][Aeea]QRP[hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F] {COOH}309 {TDA}[AC4Abu][Aeea][Aeea][hArg][hArg]Q[hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[4-Cl—F]{COOH} 310{TDA}[AC4Abu][Aeea][Aeea][hArg]Q[hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[4-Cl—F]{COOH} 311{TDA}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]P[Nle][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 312{TDA}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]Pr[NMeLeu]SH[Nle]G[Oic][pI-Phe]P[D-Bip]{COOH} 313{TDA}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]PK[NMeLeu]SH[Nle]G[Oic][pI-Phe]P[D-Bip]{COOH} 314{TDA}[AC4Abu][Aeea][Aeea][Nle]rQ[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 315{TDA}[AC4Abu][Aeea][Aeea][hArg][Nle]Q[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 316{TDA}[AC4Abu][Aeea][Aeea][hArg]rQ[Nle]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 317{TDA}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]P[Nle][NMeLeu]AHKG[Oic][pI-Phe]P[D-Bip]{COOH} 318{TDA}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]Pr[NMeLeu]AH[Nle]G[Oic][pI-Phe]P[D-Bip]{COOH} 319{TDA}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]PK[NMeLeu]AH[Nle]G[Oic][pI-Phe]P[D-Bip]{COOH} 320{TDA}[AC4Abu][Aeea][Aeea][Nle]rQ[hArg]Pr[NMeLeu]AHKG[Oic][pI-Phe]P[D-Bip]{COOH} 321{TDA}[AC4Abu][Aeea][Aeea][hArg][Nle]Q[hArg]Pr[NMeLeu]AHKG[Oic][pI-Phe]P[D-Bip]{COOH} 322{TDA}[AC4Abu][Aeea][Aeea][hArg]rQ[Nle]Pr[NMeLeu]AHKG[Oic][pI-Phe]P[D-Bip]{COOH} 323{TDA}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]P[Nle][NMeLeu]SAKG[Oic][pI-Phe]P[D-Bip]{COOH} 324{TDA}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]Pr[NMeLeu]SA[Nle]G[Oic][pI-Phe]P[D-Bip]{COOH} 325{TDA}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]PK[NMeLeu]SA[Nle]G[Oic][pI-Phe]P[D-Bip]{COOH} 326{TDA}[AC4Abu][Aeea][Aeea][Nle]rQ[hArg]Pr[NMeLeu]SAKG[Oic][pI-Phe]P[D-Bip]{COOH} 327{TDA}[AC4Abu][Aeea][Aeea][hArg][Nle]Q[hArg]Pr[NMeLeu]SAKG[Oic][pI-Phe]P[D-Bip]{COOH} 328{TDA}[AC4Abu][Aeea][Aeea][hArg]rQ[Nle]Pr[NMeLeu]SAKG[Oic] [pI-Phe]P[D-Bip]{COOH} 329{TDA}[AC4Abu][Aeea][Aeea][hArg]Q[hArg]P[Nle][NMeLeu]AAKG[Oic][pI-Phe]P[D-Bip]{COOH} 330{TDA}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]Pr[NMeLeu]AA[Nle]G[Oic][pI-Phe]P[D-Bip]{COOH} 331{TDA}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]PK[NMeLeu]AA[Nle]G[Oic][pI-Phe]P[D-Bip]{COOH} 332{TDA}[AC4Abu][Aeea][Aeea][Nle]rQ[hArg]Pr[NMeLeu]AAKG[Oic][pI-Phe]P[D-Bip]{COOH} 333{TDA}[AC4Abu][Aeea][Aeea][hArg][Nle]Q[hArg]Pr[NMeLeu]AAKG[Oic][pI-Phe]P[D-Bip]{COOH} 334{TDA}[AC4Abu][Aeea][Aeea][hArg]rQ[Nle]Pr[NMeLeu]AAKG[Oic][pI-Phe]P[D-Bip]{COOH} 335{TDA}[AC4Abu][Aeea][Aeea]RQRP[Nle][Cha]SHKG[Oic][Nle]P[4-Cl—F] {COOH}336 {TDA}[AC4Abu][Aeea][Aeea]RQRP[hArg][Cha]SH[Nle]G[Oic][Nle]P[4-Cl—F]{COOH} 337{TDA}[AC4Abu][Aeea][Aeea]RQRPK[Cha]SH[Nle]G[Oic][Nle]P[4-Cl—F] {COOH}338 {TDA}[AC4Abu][Aeea][Aeea][Nle] Q R P [hArg] [Cha] S H K G [Oic][Nle] P [4-Cl—F] {COOH} 339 {TDA}[AC4Abu][Aeea][Aeea] R Q [Nle] P [hArg][Cha] S H K G [Oic] [Nle] P [4-Cl—F] {COOH} 340{TDA}[AC4Abu][Aeea][Aeea] R Q R P [hArg] [Cha] S A [Nle] G [Oic] [Nle] P[4-Cl—F] {COOH} 341 {TDA}[AC4Abu][Aeea][Aeea] R Q R P K [Cha] S A [Nle]G [Oic] [Nle] P [4-Cl—F] {COOH} 342 {TDA}[AC4Abu][Aeea][Aeea] [Nle] Q RP [hArg] [Cha] S A K G [Oic] [Nle] P [4-Cl—F] {COOH} 343{TDA}[AC4Abu][Aeea][Aeea] R Q [Nle] P [hArg] [Cha] S A K G [Oic] [Nle] P[4-Cl—F] {COOH} 344 {TDA}[AC4Abu][Aeea][Aeea] R Q R P [Nle] [Cha] A H KG [Oic] [Nle] P [4-Cl—F] {COOH} 345 {TDA}[AC4Abu][Aeea][Aeea] R Q R P[Nle] [Cha] A A K G [Oic] [Nle] P [4-Cl—F] {COOH} 346{TDA}[AC4Abu][Aeea][Aeea] R Q R P K [Cha] A A [Nle] G [Oic] [Nle] P[4-Cl—F] {COOH} 347 {TDA}[AC4Abu][Aeea][Aeea] [Nle] Q R P [hArg] [Cha] AA K G [Oic] [Nle] P [4-Cl—F] {COOH} 348 {TDA} [AC4Abu] [Aeea] [Aeea] R Q[Nle] P [hArg] [Cha] A A K G [Oic] [Nle] P [4-Cl—F] {COOH} 349 {DDDA}[AC4Abu] [Aeea] [Aeea] Q [hArg] P r [NMeLeu] S H K G [Oic] [pI-Phe] P[D-Bip] {COOH} 350 {TDA} [AC4Abu] [Aeea] [Aeea] R Q R P [Nle] [Cha] S AK G [Oic] [Nle] P [4-Cl—F] {COOH} 351 {TDA} [AC4Abu] [Aeea] [Aeea] R Q RP [hArg] [Cha] A A [Nle] G [Oic] [Nle] P [4-Cl—F] {COOH} 352 {TDA}[AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] L S H K G [Oic] [Nle] P [4-Cl—F]{COOH} 353 {TDA} [AC4Abu] [Aeea] [Aeea] Q [hArg] P [NMeArg] L S H K G P[Nle] P [4-Cl—F] {COOH} 354 {TDA} [AC4Abu] [Aeea] [Aeea] Q [hArg] P[NMeArg] L S H K G [Oic] [Nle] P [4-Cl—F] {COOH} 355 {TDA} [AC4Abu][Aeea] [Aeea] Q R P [hArg] L S H K G P [Nle] P [4-Cl—F] {COOH} 356 {TDA}[AC4Abu] [Aeea] [Aeea] Q R P [hArg] L S H K G [Oic] [Nle] P [4- Cl—F]{COOH} 357 {TDA} [AC4Abu] [Aeea] [Aeea] Q [hArg] P [hArg] L S H K G P[Nle] P [4- Cl—F] {COOH} 358 {TDA} [AC4Abu] [Aeea] [Aeea] Q [hArg] P[hArg] L S H K G [Oic] [Nle] P [4-Cl—F] {COOH} 359 {TDA} [AC4Abu] [Aeea][Aeea] Q [hArg] P [NMeArg] [aMeLeu] S H K G [Oic] [Nle] P [4-Cl—F]{COOH} 360 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [hArg] [aMeLeu] S H K G[Oic] [Nle] P [4-Cl—F] {COOH} 361 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P[NMeArg] [Cha] S H K G P [Nle] P [4-Cl—F] {COOH} 362 {TDA} [AC4Abu][Aeea] [Aeea] Q [hArg] P [NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl—F]{COOH} 363 {TDA} [AC4Abu] [Aeea] [Aeea] q [hArg] P r [Cha] S H K G [Oic][Nle] P [4-Cl—F] {COOH} 364 {TDA} [AC4Abu] [Aeea] [Aeea] Q [hArg] P r[Cha] S H K G [Oic] [Nle] P [D-4ClF] {COOH} 365 {TDA} [AC4Abu] [Aeea][Aeea] q [hArg] P r [Cha] S H K G [Oic] [Nle] P [Oic] {COOH} 366 {TDA}[AC4Abu] [Aeea] [Aeea] q [hArg] P r [Cha] S H K G [Oic] [Nle] P [Aic]{COOH} 367 {TDA} [AC4Abu] [Aeea] [Aeea] q [hArg] P r [Cha] S H K G [Oic][Nle] P [1-Nal] {COOH} 368 {TDA} [AC4Abu] [Aeea] [Aeea] q [hArg] P r[Cha] S H K G [Oic] [Nle] P [2-Nal] {COOH} 369 {TDA} [AC4Abu] [Aeea][Aeea] q [hArg] P r [Cha] S H K G [Oic] [Nle] P [D-4IF] {COOH} 370 {TDA}[AC4Abu] [Aeea] [Aeea] q [hArg] P r [Cha] S H K G [Oic] [Nle] P [Bh4ClF]{COOH} 371 {TDA} [AC4Abu] [Aeea] [Aeea] q [hArg] P r [Cha] S H K [Tle][Oic] [Nle] P [4-Cl—F] {COOH} 372 {TDA} [AC4Abu] [Aeea] [Aeea] q [hArg]P r [Cha] S A K G [Oic] [Nle] P [4-Cl—F] {COOH} 373 {TDA} [AC4Abu][Aeea] [Aeea] q [hArg] P r [Cha] S Y K G [Oic] [Nle] P [4-Cl—F] {COOH}374 {TDA} [AC4Abu] [Aeea] [Aeea] [BLeu] R P [NMeArg] [aMeLeu] S H K G[Oic] [Nle] P [4-Cl—F] {COOH} 375 {TDA} [AC4Abu] [Aeea] [Aeea] [aMeLeu]R P [NMeArg] [aMeLeu] S H K G [Oic] [Nle] P [4-Cl—F] {COOH} 376 {TDA}[AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] [aMeLeu] S H K G [Oic] [Nle][aMePro] [4-Cl—F] {COOH} 377 {TDA} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg][Cha] S H K G [Oic] [Nle] P [4-Cl—F] {COOH} 378 {TDA} [AC4Abu] [Aeea][Aeea] q R P [hArg] [BLeu] S H K G [Oic] [Nle] P [4-Cl—F] {COOH} 379{TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] [Cha] S H K G [Oic] [Nle] P[BhPhe] {COOH} 380 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] [Cha] S HK G [Oic] [Nle] P [D-BhPhe] {COOH} 381 {TDA} [AC4Abu] [Aeea] [Aeea] Q RP [NMeArg] [Cha] S H K G [Oic] [Nle] P [D-4ClF] {COOH} 382 {TDA}[AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib][4-Cl—F] {COOH} 383 {TDA} [AC4Abu] [Aeea] [Aeea] Q [hArg] P [NMeArg][BLeu] S H K G P [Nle] P [4-Cl—F] {COOH} 384 {TDA} [AC4Abu] [Aeea][Aeea] Q r P [NMehArg] [BLeu] S H K G P [Nle] P [4-Cl—F] {COOH} 385{TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] [aMeLeu] S H K G [Oic] [Nle]P [4-Cl—F] {COOH} 386 {TDA} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg][aMeLeu] S H K G [Oic] [Nle] P [4-Cl—F] {COOH} 387 {TDA} [AC4Abu] [Aeea][Aeea] Q R P [NMeArg] [aMeLeu] S H K G [aMePro] [Nle] P [4-Cl—F] {COOH}388 {TDA} [AC4Abu] [Aeea] [Aeea] [BLeu] R P [NMeArg] [aMeLeu] S H K G[Oic] [Nle] [aMePro] [4-Cl—F] {COOH} 389 {TDA} [AC4Abu] [Aeea] [Aeea] QR P [NMeArg] [aMeLeu] S H K G [Oic] [Nle] [4-Cl—F] {COOH} 390 {TDA}[AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] [Cha] S H K G [Oic] [Nle] P[4-Cl—F] {COOH} 391 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] [BLeu] SH K G [Oic] [Nle] P [4-Cl—F] {COOH} 392 {TDA} [AC4Abu] [Aeea] [Aeea] Q R[aMePro] [hArg] [BLeu] S H K G [Oic] [Nle] P [4-Cl—F] {COOH} 393 {TDA}[AC4Abu] [Aeea] [Aeea] [BLeu] R P [NMeArg] [Cha] S H K G [Oic] [Nle] P[4-Cl—F] {COOH} 394 {TDA} [AC4Abu] [Aeea] [Aeea] [BLeu] R P [NMeArg][BLeu] S H K G [Oic] [Nle] P [4-Cl—F] {COOH} 395 {TDA} [AC4Abu] [Aeea][Aeea] [BLeu] R P [hArg] [BLeu] S H K G [Oic] [Nle] P [4-Cl—F] {COOH}396 {TDA} [AC4Abu] [Aeea] [Aeea] Q R [NMeArg] [aMeLeu] S H K G [Oic][Nle] P [4-Cl—F] {COOH} 397 {TDA} [AC4Abu] [Aeea] [Aeea] Q [NMeArg][aMeLeu] S H K G [Oic] [Nle] P [4-Cl—F] {COOH} 398 {TDA} [AC4Abu] [Aeea][Aeea] [Aeea] [Aeea] [Aeea] [Aeea] Q R P [NMeArg] L S H K G P [Nle] P[4-Cl—F] {COOH} 399 {TDA} [AC4Abu] [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg]L S H K G P [Nle] P [4-Cl—F] {COOH} 400 {TDA} [AC4Abu] [Aeea] [Aeea] Q[Cit] P [NMeArg] L S H K G P [Nle] P [4-Cl—F] {COOH} 401 {TDA} [AC4Abu][Aeea] [Aeea] Q R P [NMeArg] L S H K G P [Nle] [aMePro] [4-Cl—F] {COOH}402 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] L S H K G P [Nle] [4-Cl—F] {COOH} 403 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] L S H K G P[Nle] [2Nal] {COOH} 404 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [hArg][aMeLeu] S H K G P [Nle] P [2Nal] {COOH} 405 {TDA} [AC4Abu] [Aeea][Aeea] Q [hArg] P [NMeArg] [Cha] S H K G [Oic] [Nle] P [2Nal] {COOH} 406{TDA} [AC4Abu] [Aeea] [Aeea] q [hArg] P r [Cha] S H K G [Oic] [Nle] P[CPG] {COOH} 407 {TDA} [AC4Abu] [Aeea] [Aeea] q [hArg] P r [Cha] S H K G[Oic] [Nle] P [D-2Nal] {COOH} 408 {TDA} [AC4Abu] [Aeea] [Aeea] Q [hArg]P [NMeArg] [Cha] S H K G P [Nle] P [4-Cl—F] {COOH} 409 {TDA} [AC4Abu][Aeea] [Aeea] Q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [4-Cl—F] {COOH}410 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] [Cha] S H K G [Oic][Nle] [D-4ClF] {COOH} 411 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg][Cha] S H K G [Oic] [Nle] [Deg] [4-Cl—F] {COOH} 412 {TDA} [AC4Abu][Aeea] [Aeea] Q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [aMePro] [4-Cl—F]{COOH} 413 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] [Cha] S H K G[Oic] [Tle] P [4-Cl—F] {COOH} 414 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P[NMeArg] [Cha] S H K G [Aib] [Nle] P [4-Cl—F] {COOH} 415 {TDA} [AC4Abu][Aeea] [Aeea] Q R P [NMeArg] [Cha] S H K G [Deg] [Nle] P [4-Cl—F] {COOH}416 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] [Cha] S H K G [Pip][Nle] P [4-Cl—F] {COOH} 417 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg][Cha] S H K G [BhPro] [Nle] P [4-Cl—F] {COOH} 418 {TDA} [AC4Abu] [Aeea][Aeea] Q R P [NMeArg] [Cha] S H K G [aMePro] [Nle] P [4-Cl—F] {COOH} 419{TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] [Cha] S A K G [Oic] [Nle] P[4-Cl—F] {COOH} 420 {TDA} [AC4Abu] [Aeea] [Aeea] [BhAsn] R P [NMeArg] LS H K G P [Nle] P [4-Cl—F] {COOH} 421 {TDA} [AC4Abu] [Aeea] [Aeea][BhLeu] R P [NMeArg] L S H K G P [Nle] P [4-Cl—F] {COOH} 422 {TDA}[AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] [BhLeu] S H K G P [Nle] P [4-Cl—F]{COOH} 423 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] L S [bAla] K G P[Nle] P [4-Cl—F] {COOH} 424 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg]L S H [bAla] G P [Nle] P [4-Cl—F] {COOH} 425 {TDA} [AC4Abu] [Aeea][Aeea] Q R P [NMeArg] L S H K G P [Nle] [bAla] [4-Cl—F] {COOH} 426 {TDA}[AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] L S H K G P [Nle] [BhPro] [4-Cl—F]{COOH} 427 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P P L S H K G P [Nle] P[4-Cl—F] {COOH} 428 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [Nle] L S H K G P[Nle] P [4-Cl—F] {COOH} 429 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg]L S H K G P [Nle] [Pip] [4-Cl—F] {COOH} 430 {TDA} [AC4Abu] [Aeea] [Aeea]Q r P r [Cha] S H K G [Oic] [Nle] P [4-Cl— F] {COOH} 431 {TDA} [AC4Abu][Aeea] [Aeea] q [hArg] P r [Cha] S H K G [Oic] [Nle] P [D-Igl] {COOH}432 {TDA} [AC4Abu] [Aeea] [Aeea] q [hArg] P r [Cha] S H K G [Oic] [Nle]P [B4ClF] {COOH} 433 {TDA} [AC4Abu] [Aeea] [Aeea] q [hArg] P r [Tle] S HK G [Oic] [Nle] P [4-Cl—F] {COOH} 434 {TDA} [AC4Abu] [Aeea] [Aeea] q[hArg] P r [Deg] S H K G [Oic] [Nle] P [4-Cl—F] {COOH} 435 {TDA}[AC4Abu] [Aeea] [Aeea] Q r P [NMehArg] [Cha] S H K G P [Nle] P [4-Cl—F]{COOH} 436 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] [Cha] S Y K G[Oic] [Nle] P [4-Cl—F] {COOH} 437 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P[NMeArg] [Cha] S V K G [Oic] [Nle] P [4-Cl—F] {COOH} 438 {TDA} [AC4Abu][Aeea] [Aeea] Q R P [NMeArg] [Cha] S [Tle] K G [Oic] [Nle] P [4-Cl—F]{COOH} 439 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] [Tle] S H K G[Oic] [Nle] P [4-Cl—F] {COOH} 440 {TDA} [AC4Abu] [Aeea] [Aeea] Q R [Pip][NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl—F] {COOH} 441 {TDA} [AC4Abu][Aeea] [Aeea] Q R [BhPro] [NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl—F]{COOH} 442 {TDA} [AC4Abu] [Aeea] [Aeea] [Tle] R P [NMeArg] [Cha] S H K G[Oic] [Nle] P [4-Cl—F] {COOH} 443 {TDA} [AC4Abu] [Aeea] [Aeea] [Deg] R P[NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl—F] {COOH} 444 {TDA} [AC4Abu][Aeea] [Aeea] Q R P [NMeArg] [Cha] S H [AMe-K] G [Oic] [Nle] P [4-Cl—F]{COOH} 445 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] [Cha] S H K G[Oic] [Nle] [Pip] [4-Cl—F] {COOH} 446 {TDA} [AC4Abu] [Aeea] [Aeea] Q R[NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl—F] {COOH} 447 {TDA} [AC4Abu][Aeea] [Aeea] Q R [NMeArg] [BLeu] S H K G [Oic] [Nle] P [4-Cl—F] {COOH}448 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] L S H H G P [Nle] P [4-Cl—F] {COOH} 449 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] L S H[1-Nal] G P [Nle] P [4-Cl—F] {COOH} 450 {TDA} [AC4Abu] [Aeea] [Aeea] Q RP [NMeArg] L [Pra] H K G P [Nle] P [4-Cl—F] {COOH} 451 {TDA} [AC4Abu][Aeea] [Aeea] q [hArg] P r [Cha] S [Deg] K G [Oic] [Nle] P [4-Cl—F]{COOH} 452 {TDA} [AC4Abu] [Aeea] [Aeea] q [hArg] P r [Cha] S [Tle] K G[Oic] [Nle] P [4-Cl—F] {COOH} 453 {TDA} [AC4Abu] [Aeea] [Aeea] q [hArg]P r [Cha] S H K G [Oic] [Nle] [D- 4IF] {COOH} 454 {TDA} [AC4Abu] [Aeea][Aeea] q [hArg] [Aib] r [Cha] S H K G [Oic] [Nle] P [4-Cl—F] {COOH} 455{TDA} [AC4Abu] [Aeea] [Aeea] q [hArg] [Deg] r [Cha] S H K G [Oic] [Nle]P [4-Cl—F] {COOH} 456 {TDA} [AC4Abu] [Aeea] [Aeea] Q r P [NMehArg][aMeLeu] S H K G P [Nle] P [4-Cl—F] {COOH} 457 {TDA} [AC4Abu] [Aeea][Aeea] Q R P [NMeArg] [aMeLeu] S H K G P [Nle] P [4-Cl—F] {COOH} 458{TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] [BLeu] S H K G P [Nle] P[4-Cl—F] {COOH} 459 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] [NMeLeu]S H K G P [Nle] P [4-Cl—F] {COOH} 460 {TDA} [AC4Abu] [Aeea] [Aeea] Q[hArg] P [NMeArg] [aMeLeu] S H K G P [Nle] P [4-Cl—F] {COOH} 461 {AdpA}[AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] L S H K G P [Nle] P [4- Cl—F]{COOH} 462 {TDA} [AC4Abu] [AC4Abu] [Aeea] Q R P [NMeArg] L S H K G P[Nle] P [4-Cl—F] {COOH} 463 {TDA} [AC4Abu] [Aeea] [Aeea] [BhGln] R P[NMeArg] L S H K G P [Nle] P [4-Cl—F] {COOH} 464 {TDA} [AC4Abu] [Aeea][Aeea] Q R P [NMeArg] L [bAla] H K G P [Nle] P [4-Cl—F] {COOH} 465 {TDA}[AC4Abu] [Aeea] [Aeea] Q R P Q L S H K G P [Nle] P [4-Cl—F] {COOH} 466{TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] L S H [Nle] G P [Nle] P[4-Cl—F] {COOH} 467 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] L[NhSerG] H K G P [Nle] P [4-Cl—F] {COOH} 468 {TDA} [AC4Abu] [Aeea][Aeea] Q R P [NMeArg] L [aMeS] H K G P [Nle] P [4-Cl—F] {COOH} 469 {TDA}[AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] L S [rHis] K G P [Nle] P [4-Cl—F]{COOH} 470 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] L S H K G P [Nle]P [2Nal] {COOH} 471 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMehArg] L S H KG P [Nle] P [2Nal] {COOH} 472 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P[NMeArg] [aMeLeu] S H K G P [Nle] P [2Nal] {COOH} 473 {TDA} [AC4Abu][Aeea] [Aeea] Q [hArg] P [NMeArg] [aMeLeu] S H K G P [Nle] P [2Nal]{COOH} 474 {Sebacicacid} [AC4Abu] [Aeea] [Aeea] [hArg] Q [hArg] P[NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl—F] {COOH} 475 {TDA} [AC4Abu][Aeea] [Aeea] Q E P [NMeArg] L S H K G P [Nle] P [4- Cl—F] {COOH} 476{TDA} [AC4Abu] [Aeea] [Aeea] Q R E [NMeArg] L S H K G P [Nle] P [4-Cl—F] {COOH} 477 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P E L S H K G P [Nle]P [4-Cl—F] {COOH} 478 {TDA} [AC4Abu] [Aeea] [Aeea] q [hArg] P [NMeArg][Cha] S H K G [Oic] [Nle] P [4-Cl—F] {COOH} 479 {TDA} [AC4Abu] [Aeea][Aeea] r Q [hArg] P [NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl—F] {COOH}480 {TDA} [AC4Abu] [Aeea] [Aeea] r Q [hArg] P [hArg] [Cha] S H K G [Oic][Nle] P [4-Cl—F] {COOH} 481 {TDA} [AC4Abu] [Aeea] [Aeea] r q [hArg] P[hArg] [Cha] S H K G [Oic] [Nle] P [4-Cl—F] {COOH} 482 {TDA} [AC4Abu][Aeea] [Aeea] Q R [aMePro] [hArg] [aMeLeu] S H K G P [Nle] P [4-Cl—F]{COOH} 483 {TDA} [AC4Abu] [Aeea] [Aeea] Q [hArg] P [NMehArg] L S H K G P[Nle] P [4-Cl—F] {COOH} 484 {TDA} [AC4Abu] [Aeea] [Aeea] Q [NMehArg] P[NMeArg] [aMeLeu] S H K G P [Nle] P [4-Cl—F] {COOH} 485 {TDA} [AC4Abu][Aeea] [Aeea] Q r P [hArg] [aMeLeu] S H K G P [Nle] P [4-Cl—F] {COOH}486 {TDA} [AC4Abu] [Aeea] [Aeea] q [hArg] [Oic] r [Cha] S H K G [Oic][Nle] P [4-Cl—F] {COOH} 487 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg][Cha] S H K G [Oic] [Nle] [BhPro] [4-Cl—F] {COOH} 488 {TDA} [AC4Abu][Aeea] [Aeea] q [hArg] P [hArg] [Cha] S H K G [Oic] [Nle] P [4-Cl—F]{COOH} 489 {TDA} [AC4Abu] [Aeea] [Aeea] Q [hArg] P [hArg] [Cha] S H K G[Oic] [Nle] P [4-Cl—F] {COOH} 490 {TDA} [AC4Abu] [Aeea] [Aeea] [hArg] Q[hArg] P [hArg] [Cha] S H K G [Oic] [Nle] P [4-Cl—F] {COOH} 491{Butanoyl} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] L S H K G P [Nle] P[4-Cl—F] {COOH} 492 {hexanoyl} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] L SH K G P [Nle] P [4-Cl—F] {COOH} 493 {Oc} [AC4Abu] [Aeea] [Aeea] Q R P[NMeArg] L S H K G P [Nle] P [4-Cl— F] {COOH} 494 {Decanoyl} [AC4Abu][Aeea] [Aeea] Q R P [NMeArg] L S H K G P [Nle] P [4-Cl—F] {COOH} 495{PDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] L S H K G P [Nle] P [4-Cl—F] {COOH} 496 {HDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] L S H K G P[Nle] P [4- Cl—F] {COOH} 497 {Succinicacid} [AC4Abu] [Aeea] [Aeea] Q R P[NMeArg] L S H K G P [Nle] P [4-Cl—F] {COOH} 498 {TDA} [AC4Abu] [Aeea][Aeea] q R P [NMeArg] [Cha] S H K G [aMePro] [Nle] [Aib] [4-Cl—F] {COOH}499 {TDA} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G [Pip][Nle] [Aib] [4-Cl—F] {COOH} 500 {TDA} [AC4Abu] [Aeea] [Aeea] q R P[NMeArg] [Cha] [BhSer] H K G [Oic] [Nle] [Aib] [4-Cl—F] {COOH} 501 {TDA}[AC4Abu] [Aeea] [Aeea] q R [aMePro] [hArg] [BLeu] S H K G [Pip] [Nle][4-Cl—F] {COOH} 502 {TDA} [AC4Abu] [Aeea] [Aeea] q R [aMePro] [hArg][BLeu] S H K G [Oic] [Nle] [aMePro] [4-Cl—F] {COOH} 503 {TDA} [AC4Abu][Aeea] [Aeea] q R [aMePro] [hArg] [BLeu] S H K G [Pip] [Nle] [aMePro][4-Cl—F] {COOH} 504 {TDA} [AC4Abu] [Aeea] [Aeea] q R [aMePro] [hArg][BLeu] S H K G [aMePro] [Nle] [aMePro] [4-Cl—F] {COOH} 505 {TDA}[AC4Abu] [Aeea] [Aeea] q R [aMePro] [hArg] [BLeu] S H K G [Oic] [Nle][Aib] [4-Cl—F] {COOH} 506 {hexanoyl} [AC4Abu] [Aeea] [Aeea] [Aeea][hArg] Q [hArg] P [NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl—F] {COOH}507 {TDA} [Aeea] [Aeea] Q R P [NMeArg] L S H K G P [Nle] P [4-Cl—F]{COOH} 508 {Subericacid} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] L S H K GP [Nle] P [4-Cl—F] {COOH} 509 {Sebacicacid} [AC4Abu] [Aeea] [Aeea] Q R P[NMeArg] L S H K G P [Nle] P [4-Cl—F] {COOH} 510 {DDDA} [AC4Abu] [Aeea][Aeea] Q R P [NMeArg] L S H K G P [Nle] P [4-Cl—F] {COOH} 511 {TetDDA}[AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] L S H K G P [Nle] P [4-Cl—F]{COOH} 512 {HexDDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] L S H K G P[Nle] P [4-Cl—F] {COOH} 513 {ODDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg]L S H K G P [Nle] P [4-Cl—F] {COOH} 514 {hexanoyl} [AC4Abu] [Aeea][Aeea] Q r P [hArg] [aMeLeu] S H K G P [Nle] P [4-Cl—F] {COOH} 515{AdpA} [AC4Abu] [Aeea] [Aeea] Q r P [hArg] [aMeLeu] S H K G P [Nle] P[4-Cl—F] {COOH} 516 {TDA} [AC4Abu] [Aeea] [Aeea] q R [BhPro] [NMeArg][Cha] S H K G [Oic] [Nle] [Aib] [4-Cl—F] {COOH} 517 {TDA} [AC4Abu][Aeea] [Aeea] q R [Pip] [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib][4-Cl—F] {COOH} 518 {TDA} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [aMeLeu][BhSer] H K G [Oic] [Nle] [aMePro] [4-Cl—F] {COOH} 519 {TDA} [AC4Abu][Aeea] [Aeea] q R [aMePro] [hArg] [BLeu] S H K G [Oic] [Nle] [4-Cl—F]{COOH} 520 {TDA} [AC4Abu] [Aeea] [Aeea] Q P [NMeArg] [Cha] S H K G [Oic][Nle] [Aib] [4-Cl—F] {COOH} 521 {TDA} [AC4Abu] [Aeea] [Aeea] q R [Pip][hArg] [BLeu] S H K G [Oic] [Nle] [Aib] [4-Cl—F] {COOH} 522 {TDA}[AC4Abu] [Aeea] [Aeea] q R [BhPro] [hArg] [BLeu] S H K G [Oic] [Nle][Aib] [4-Cl—F] {COOH} 523 {TDA} [AC4Abu] [Aeea] [Aeea] q R [aMePro][Nle] [BLeu] S H K G [Oic] [Nle] [aMePro] [4-Cl—F] {COOH} 524 {TDA}[AC4Abu] [Aeea] [Aeea] q E P [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib][4-Cl—F] {COOH} 525 {TDA} [AC4Abu] [Aeea] [Aeea] q e P [NMeArg] [Cha] SH K G [Oic] [Nle] [Aib] [4-Cl—F] {COOH} 526 {TDA} [AC4Abu] [Aeea] [Aeea]q R P E [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl—F] {COOH} 527 {TDA}[AC4Abu] [Aeea] [Aeea] q R P e [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl—F]{COOH} 528 {TDA} [AC4Abu] [Aeea] [Aeea] q R P [Nle] [Cha] S H K G [Oic][Nle] [Aib] [4-Cl—F] {COOH} 529 {TDA} [AC4Abu] [Aeea] [Aeea] q R P [Nle][Cha] S A K G [Oic] [Nle] [Aib] [4-Cl—F] {COOH} 530 {TDA} [AC4Abu][Aeea] [Aeea] q R P [NMeArg] [Cha] S A K G [Oic] [Nle] [Aib] [4-Cl—F]{COOH} 531 {TDA} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S Y K G[Oic] [Nle] [Aib] [4-Cl—F] {COOH} 532 {TDA} [AC4Abu] q R P [NMeArg][Cha] S H K G [Oic] [Nle] [Aib] [4-Cl— F] {COOH} 533 {TDA} [AC4Abu][AC4Abu] q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl—F] {COOH}534 {TDA} [AC4Abu] [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G[Oic] [Nle] [Aib] [4-Cl—F] {COOH} 535 {TDA} [AC4Abu] [AC4Abu] [Aeea] q RP [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl—F] {COOH} 536 {TDA}[AC4Abu] [Aeea] [AC4Abu] q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib][4-Cl—F] {COOH} 537 {TDA} E [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G[Oic] [Nle] [Aib] [4-Cl—F] {COOH} 538 {TDA} d [Aeea] [Aeea] q R P[NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl—F] {COOH} 539 {TDA}[AC4Abu] [NPeg11] q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib][4-Cl—F] {COOH} 540 {TDA} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] SH K G [Oic] [Nle] [Aib] [4-Cl—F] {COOH} 541 {TDA} [AC4Abu] [Aeea] [Aeea]q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [aMePro] [4-Cl—F] {COOH} 542{TDA} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G [Oic] [Nle][BhPro] [4-Cl—F] {COOH} 543 {TDA} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg][Cha] S H K [Tle] [Oic] [Nle] [Aib] [4-Cl—F] {COOH} 544 {TDA} [AC4Abu][Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G [Pip] [Nle] [BhPro] [4-Cl—F]{COOH} 545 {TDA} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G[aMePro] [Nle] [BhPro] [4-Cl—F] {COOH} 546 {TDA} D [Aeea] [Aeea] q R P[NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl—F] {COOH} 547 {TDA} e[Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl—F]{COOH} 548 {H2} H A E G T F T S D V S S Y L E G Q A A K(AC4Abu-Palm) E FI A W L V R G R G {COOH} 549 {TDA} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg][Cha] S H K G [Oic] [Nle] [Deg] [4-Cl—F] {COOH} 550 {TDA} [AC4Abu][Aeea] [Aeea] Q [BhPro] [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib][4-Cl—F] {COOH} 551 {TDA} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] SH K G [aMePro] [Nle] [4-Cl—F] {COOH} 552 {TDA} [AC4Abu] [Aeea] [Aeea][BLeu] R [aMePro] [hArg] [aMeLeu] S H K G [Oic] [Nle] [aMePro] [4-Cl—F]{COOH} 553 {TDA} [AC4Abu] [Aeea] [Aeea] q R [BhPro] [NMeArg] [aMeLeu] SH K G [Oic] [Nle] [aMePro] [4-Cl—F] {COOH} 554 {TDA} [AC4Abu] [Aeea][Aeea] q R P [NMeArg] [aMeLeu] S H K G [Oic] [Nle] [BhPro] [4-Cl—F]{COOH} 555 {TDA} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [aMeLeu] S H K G[Pip] [Nle] [aMePro] [4-Cl—F] {COOH} 556 {TDA} [AC4Abu] [Aeea] [Aeea] qR [aMePro] [hArg] [BLeu] S H K G [Oic] [Nle] [BhPro] [4-Cl—F] {COOH} 557{TDA} [AC4Abu] [Aeea] [Aeea] [Tle] [hArg] P r [Cha] S H K G [Oic] [Nle]P [4-Cl—F] {COOH} 558 {TDA} [AC4Abu] [Aeea] [Aeea] [Deg] [hArg] P r[Cha] S H K G [Oic] [Nle] P [4-Cl—F] {COOH} 559 {hexanoyl} [AC4Abu][Aeea] [Aeea] Q R P [hArg] [aMeLeu] S H K G P [Nle] P [4-Cl—F] {COOH}560 {hexanoyl} [AC4Abu] [Aeea] [Aeea] Q R [aMePro] [hArg] [aMeLeu] S H KG P [Nle] P [4-Cl—F] {COOH} 561 {hexanoyl} [AC4Abu] [Aeea] [Aeea] Q[NMehArg] P [NMeArg] [aMeLeu] S H K G P [Nle] P [4-Cl—F] {COOH} 562{hexanoyl} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] [Cha] S H K G [Oic][Nle] [Aib] [4-Cl—F] {COOH} 563 {hexanoyl} [AC4Abu] [Aeea] [Aeea] [hArg]r Q [hArg] P r [NMeLeu] S H K G [Pip] [pI-Phe] P [D-Bip] {COOH} 564{TDA} [AC4Abu] [Aeea] [Aeea] R Q [Nle] P [hArg] [Cha] S A K G [Pip][Nle] P [4-Cl—F] {COOH} 565 {TDA} [AC4Abu] [Aeea] [Aeea] r Q R P [hArg][NMeLeu] S H K G [Pip] [pI-Phe] P [D-Bip] {COOH} 566 {hexanoyl} [Ahx][AC4Abu] [Aeea] [Aeea] [hArg] r Q [hArg] P r [NMeLeu] S H K G [Oic][pI-Phe] P [D-Bip] {COOH} 567 {Palm} [AC4Abu] [Aeea] [Aeea] q R P[NMeArg] [Cha] S H K G [Oic] [Nle] P [D-BhPhe] {COOH} 568 {Palm}[AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G [Pip] [Nle] P[D-BhPhe] {COOH} 569 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha]S H K G [Oic] [Nle] [Aib] [D-BhPhe] {COOH} 570 {Palm} [AC4Abu] [Aeea][Aeea] q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [aMePro] [D-BhPhe]{COOH} 571 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G[Oic] [Nle] [BhPro] [D-BhPhe] {COOH} 572 {Palm} [AC4Abu] [Aeea] [Aeea] qR P [NMeArg] [Cha] S H K G [Oic] [Nle] [bAla] [D-BhPhe] {COOH} 573{Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G [Oic] [Nle][D-BhPhe] {COOH} 574 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha]S H K G [Pip] [Nle] [D-BhPhe] {COOH} 575 {Palm} [AC4Abu] [Aeea] [Aeea] qR P [NMeArg] [Cha] S H K G [Oic] [Nle] P [BhPhe] {COOH} 576 {HDA}[AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G [Oic] [Nle] P[D-4ClF] {COOH} 577 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] SH K G [Oic] [Nle] [Aib] [BhPhe] {COOH} 578 {Palm} [AC4Abu] [Aeea] [Aeea]q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [BhPhe] {COOH} 579 {Palm}[AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G [Pip] [Nle] [BhPhe]{COOH} 580 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G[Oic] [Aib] [4-Cl—F] {COOH} 581 {Palm} [AC4Abu] [Aeea] [Aeea] q R P[NMeArg] [Cha] S H K G [Pip] [Aib] [4-Cl—F] {COOH} 582 {Palm} [AC4Abu][Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G [Nip] [Nle] [Aib] [4-Cl—F]{COOH} 583 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G[Inp] [Nle] [Aib] [4-Cl—F] {COOH} 584 {Palm} [AC4Abu] [Aeea] [Aeea] q RP [NMeArg] [Cha] S H K G [Oic] [Nle] [1-Ach] [4-Cl—F] {COOH} 585 {Palm}[AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Acp][4-Cl—F] {COOH} 586 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] SH K G [Oic] [Nle] [Nip] [4-Cl—F] {COOH} 587 {Palm} [AC4Abu] [Aeea][Aeea] q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Inp] [4-Cl—F] {COOH}588 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G [Pip][Nle] [Nip] [4-Cl—F] {COOH} 589 {Palm} [AC4Abu] [Aeea] [Aeea] Q R P[NMeArg] [Cha] S H K G [Pip] [Nle] [Inp] [4-Cl—F] {COOH} 590 {Palm}[AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G [Pip] [Nle] P [Aic]{COOH} 591 {Dodecanoyl-NH} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] L S H KG P [Nle] P [4-Cl—F] {COOH} 592 {Myristyl-NH} [AC4Abu] [Aeea] [Aeea] Q RP [NMeArg] L S H K G P [Nle] P [4-Cl—F] {COOH} 593 {Palm} [AC4Abu][Aeea] [Aeea] Q R P [NMeArg] L S H K G P [Nle] P [4- Cl—F] {COOH} 594{St} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] L S H K G P [Nle] P [4-Cl— F]{COOH} 595 {TDA} [AC4Abu] [Aeea] [Aeea] q [hArg] P r [Cha] S H K G [Oic][Nle] [Aic] {COOH} 596 {TDA} [AC4Abu] [Aeea] [Aeea] q [hArg] P r [Cha] SH K G [Oic] [Nle] [Oic] {COOH} 597 {Palm} [AC4Abu] [Aeea] [Aeea] q R P[NMeArg] [Cha] S H K G [Pip] [Nle] [Aic] {COOH} 598 {Palm} [AC4Abu][Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G [Pip] [Nle] P [Tic] {COOH}599 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G [Pip][Nle] P [D-Tic] {COOH} 600 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg][Cha] S H K G [Oic] [Nle] [BhPro] [D-4ClF] {COOH} 601 {Palm} [AC4Abu][Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Pip] [D-4ClF]{COOH} 602 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G[Oic] [Nle] [Nip] [D-4ClF] {COOH} 603 {Palm} [AC4Abu] [Aeea] [Aeea] q RP [NMeArg] [Cha] S H K G [Oic] [Nle] [Inp] [D-4ClF] {COOH} 604 {Palm}[AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib][D-4ClF] {COOH} 605 {TDA} [AC4Abu] [Aeea] [Aeea] Q R P [NMeArg] [aMeLeu]S H K G [Pip] [Nle] [aMePro] [4-Cl—F] {COOH} 606 {Palm} [AC4Abu] [Aeea][Aeea] q R P [NMeArg] [Cha] S H K G [Oic] [Nle] P [AMEF] {COOH} 607{Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G [Oic] [Nle] P[D-AMF] {COOH} 608 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] SH K G [Oic] [Nle] [Aib] [AMEF] {COOH} 609 {Palm} [AC4Abu] [Aeea] [Aeea]q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Pip] [AMEF] {COOH} 610 {Palm}[AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [aMePro][AMEF] {COOH} 611 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S HK G [Oic] [Nle] [BhPro] [AMEF] {COOH} 612 {Palm} [AC4Abu] [Aeea] [Aeea]q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [BhPro] [D-AMF] {COOH} 613{Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G [BhPro] [Nle][4-Cl—F] {COOH} 614 {Palm} [AC4Abu] [Aeea] [Aeea] q [hArg] P r [Cha] S HK G [Oic] [Nle] P [D-BhPhe] {COOH} 615 {Palm} [AC4Abu] [Aeea] [Aeea] q RP [NMeArg] [Cha] [BLeu] H K G [Oic] [Nle] [BhPro] [D-BhPhe] {COOH} 616{Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S [Dap] K G [Oic][Nle] [BhPro] [D-BhPhe] {COOH} 617 {Palm} [AC4Abu] [Aeea] [Aeea] q R P[NMeArg] [Cha] S h K G [Oic] [Nle] [BhPro] [D-BhPhe] {COOH} 618 {Palm}[AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S H O G [Oic] [Nle] [BhPro][D-BhPhe] {COOH} 619 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha]S H [aMeOrn] G [Oic] [Nle] [BhPro] [D-BhPhe] {COOH} 620 {Palm} [AC4Abu][Aeea] [Aeea] q R P [NMeArg] [Cha] S H [3Pal] G [Oic] [Nle] [BhPro][D-BhPhe] {COOH} 621 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha]S H [4-Cl—F] G [Oic] [Nle] [BhPro] [D-BhPhe] {COOH} 622 {Palm} [AC4Abu][Aeea] [Aeea] q R P [NMeArg] [Cha] S H [4-F—F] G [Oic] [Nle] [BhPro][D-BhPhe] {COOH} 623 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha]S H [Dab] G [Oic] [Nle] [BhPro] [D-BhPhe] {COOH} 624 {Palm} [AC4Abu][Aeea] [Aeea] q R P [NMeArg] [Cha] S H [Dap] G [Oic] [Nle] [BhPro][D-BhPhe] {COOH} 625 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha]S H [Igl] G [Oic] [Nle] [BhPro] [D-BhPhe] {COOH} 626 {Palm} [AC4Abu][Aeea] [Aeea] q R P [NMeArg] [Cha] S H K [Sar] [Oic] [Nle] [BhPro][D-BhPhe] {COOH} 627 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg][aMeLeu] [aMeS] H K G [Oic] [Nle] [BhPro] [4-Cl—F] {COOH} 628 {Palm}[AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [aMeLeu] [BLeu] H K G [Oic] [Nle][BhPro] [4-Cl—F] {COOH} 629 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg][aMeLeu] S [Bip] K G [Oic] [Nle] [BhPro] [4-Cl—F] {COOH} 630 {Palm}[AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [aMeLeu] S [Dap] K G [Oic] [Nle][BhPro] [4-Cl—F] {COOH} 631 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg][aMeLeu] S h K G [Oic] [Nle] [BhPro] [4-Cl—F] {COOH} 632 {Palm} [AC4Abu][Aeea] [Aeea] q R P [NMeArg] [aMeLeu] S H [NPipG] G [Oic] [Nle] [BhPro][4-Cl—F] {COOH} 633 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] SH [NPipG] G [Oic] [Nle] [BhPro] [D-BhPhe] {COOH} 634 {Palm} [AC4Abu][Aeea] [Aeea] q R P [NMeArg] [aMeLeu] S H O G [Oic] [Nle] [BhPro][4-Cl—F] {COOH} 635 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg][aMeLeu] S H [aMeOrn] G [Oic] [Nle] [BhPro] [4-Cl—F] {COOH} 636 {Palm}[AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [aMeLeu] S H [3Pal] G [Oic] [Nle][BhPro] [4-Cl—F] {COOH} 637 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg][aMeLeu] S H [4-F—F] G [Oic] [Nle] [BhPro] [4-Cl—F] {COOH} 638 {Palm}[AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [aMeLeu] S H [Dab] G [Oic] [Nle][BhPro] [4-Cl—F] {COOH} 639 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg][aMeLeu] S H [Dap] G [Oic] [Nle] [BhPro] [4-Cl—F] {COOH} 640 {Palm}[AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [aMeLeu] S H [Igl] G [Oic] [Nle][BhPro] [4-Cl—F] {COOH} 641 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg][aMeLeu] S H K [Sar] [Oic] [Nle] [BhPro] [4-Cl—F] {COOH} 642 {Palm}[AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] [aMeS] H K G [Oic] [Nle][BhPro] [D-BhPhe] {COOH} 643 {Palm} [AC4Abu] [Aeea] [Aeea] q R P[NMeArg] [Cha] [NPrG] H K G [Pip] [Nle] [Aib] [4-Cl—F] {COOH} 644 {Palm}[AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] S [NPrG] K G [Pip] [Nle][Aib] [4-Cl—F] {COOH} 645 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NPipG][Cha] S H K G [Pip] [Nle] [Aib] [4-Cl—F] {COOH} 646 {Palm} [AC4Abu][Aeea] [Aeea] q [NPipG] P [NMeArg] [Cha] S H K G [Pip] [Nle] [Aib][4-Cl—F] {COOH} 647 {Palm} [AC4Abu] [Aeea] [Aeea] q R P [NMeArg] [Cha] SH K G [Pip] [Nle] P [BhPhe] {COOH}

Any of fatty acyl groups in the specific peptide conjugates listed inTable 7 can be substituted with another fatty acyl group, such as thoselisted in Table 6. By way of example, a TDA fatty acyl group in any ofthe specific peptides listed above can be substituted with an HDA, ODDA,or other fatty acyl group. In some embodiments, a fatty acyl group witha shorter carbon chain may be substituted for a fatty acyl group with alonger carbon chain in the peptide-conjugates listed above. Forinstance, a butanoyl or hexanoyl group may be substituted for apalmitoyl, TDA, HDA, or ODDA fatty acyl group. In other embodiments, thefatty acyl group in the specific peptide conjugates listed in Table 7may be substituted with another half-life extending moiety (e.g., PEG oran immunoglobulin Fc) as described herein. In alternative embodiments,any of the apelin peptide portions of the conjugates may be used as APJagonists in an unconjugated form.

In certain embodiments, the half-life extension moiety or vehicle is apolymer. Thus, any of the modified apelin polypeptides disclosed hereincan be conjugated, optionally through a conjugation linker, to apolymer, particularly a water-soluble polymer. In some embodiments, thepolymer half-life extending moiety is a polyethylene glycol (PEG)polymer, covalently linked, optionally through a conjugation linker, atthe N-terminus, C-terminus or at one or more intercalary side chains ofthe apelin polypeptide. In some embodiments, the peptide conjugate mayinclude one or more PEG moieties conjugated to a non-PEG half-lifeextending moiety or to the apelin polypeptide, or to any combination ofany of these. For example, an immunoglobulin or immunoglobulin Fc domainor portion thereof can be made mono-PEGylated, di-PEGylated, orotherwise multi-PEGylated, by the process of covalent conjugation, andthe apelin polypeptide may conjugated to the immunoglobulin orimmunoglobulin Fc domain via the one or more PEG moieties.

Covalent conjugation of proteins and peptides with PEG can significantlyextend the in vivo circulating half-lives. PEGylation is believed toachieve this effect predominately by retarding renal clearance, sincethe PEG moiety adds considerable hydrodynamic radius to the molecule(see Zalipsky et al., “Use of functionalized poly(ethylene glycol)s formodification of polypeptides, in poly(ethylene glycol) chemistry:Biotechnical and biomedical applications”, J. M. Harris, Ed., PlenumPress: New York, 347-370, 1992). Additional benefits often conferred byPEGylation of proteins and peptides include increased solubility,resistance to proteolytic degradation, and reduced immunogenicity of thetherapeutic polypeptide. The merits of protein PEGylation are evidencedby the commercialization of several PEGylated proteins includingPEG-Adenosine deaminase (Adagen™/Enzon Corp.), PEG-L-asparaginase(Oncaspar™/Enzon Corp.), PEG-Interferon α-2b(PEG-Intron™/Schering/Enzon), PEG-Interferon α-2a (PEGASYS™/Roche) andPEG-G-CSF (Neulasta™/Amgen) as well as many others in clinical trials.

By “PEGylated peptide,” “PEGylated polypeptide” or “PEGylated protein”is meant a peptide having a polyethylene glycol (PEG) moiety covalentlybound to an amino acid residue of the peptide itself or to a peptidyl ornon-peptidyl linker that is covalently bound to a residue of thepeptide, either directly or indirectly through another linker moiety. Anon-limiting example is N-terminal conjugation of the peptide with3-(1-(1-bromo-2-oxo-6,9,12,15,18,21,24,27,30,33,36-undecaoxa-3-azaoctatriacontan-38-yl)-1H-1,2,3-triazol-4-yl)propanoyl(designated herein by the abbreviation“{bromoacetamide-PEG11-triazole}-”).

By “polyethylene glycol” or “PEG” is meant a polyalkylene glycolcompound or a derivative thereof, with or without coupling agents orderivatization with coupling or activating moieties (e.g., withaldehyde, hydroxysuccinimidyl, hydrazide, thiol, triflate, tresylate,azirdine, oxirane, orthopyridyl disulphide, vinylsulfone, iodoacetamideor a maleimide moiety). In accordance with various embodiments, usefulPEG includes substantially linear, straight chain PEG, branched PEG(brPEG), or dendritic PEG. See, e.g., U.S. Pat. Nos. 5,171,264;5,932,462; 6,602,498.

Briefly, the PEG groups can generally be attached to the peptide portionof the composition via acylation or alkylation (or reductive amination)through a reactive group on the PEG moiety (e.g., an aldehyde, amino,thiol, or ester group) to a reactive group on the compound (e.g., analdehyde, amino, or ester group). A useful strategy for the PEGylationof synthetic peptides consists of combining, through forming a conjugatelinkage in solution, a peptide and a PEG moiety, each bearing a specialfunctionality that is mutually reactive toward the other. The peptidescan be easily prepared with conventional solid phase synthesis. Thepeptides can be “preactivated” with an appropriate functional group at aspecific site. The precursors are purified and fully characterized priorto reacting with the PEG moiety. Ligation of the peptide with PEGusually takes place in aqueous phase and can be easily monitored byreverse phase analytical HPLC. The PEGylated peptides can be easilypurified by preparative HPLC and characterized by analytical HPLC, aminoacid analysis and laser desorption mass spectrometry.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, 3:138-161). In theapplication, the term “PEG” is used broadly to encompass anypolyethylene glycol molecule, in mono-, bi-, or poly-functional form,without regard to size or to modification at an end of the PEG, and canbe represented by the formula:X—O(CH₂CH₂O)_(n)—R,where n is 3 to 2300 and X is H or a terminal modification, e.g., amethyl or C₁₋₄ alkyl, and R is the reactive moiety used for covalentattachment.

In some embodiments, a PEG can be used that terminates on one end withhydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). It is notedthat the other end of the PEG, which is shown in the formula aboveterminating in R, covalently attaches to an activating moiety via anether oxygen bond, an amine linkage, or amide linkage. When used in achemical structure, the term “PEG” includes the formula above withoutthe hydrogen of the hydroxyl group shown, leaving the oxygen availableto react with a free carbon atom of a linker to form an ether bond. Morespecifically, in order to conjugate PEG to a peptide, the peptide mustbe reacted with PEG in an “activated” form. Activated PEG can berepresented by the formula:(PEG)-(A)

where PEG (defined supra) covalently attaches to a carbon atom of theactivation moiety (A) to form an ether bond, an amine linkage, or amidelinkage, and (A) contains a reactive group which can react with anamino, azido, alkyne, imino, maleimido, N-succinimidyl, carboxyl,aminooxy, seleno, or thiol group on an amino acid residue of a peptideor a linker moiety covalently attached to the apelin peptide.

Examples of various PEG moieties are shown below and in FIG. 2 .

Aeaa ([2-(2-Amino-ethoxy)-ethoxy]-acetic acid) is another PEG that canbe used to increase stability of apelin peptides or can serve a spatial,functional, and/or structural role in a conjugation linker of the apelinpolypeptide. The structure of Aeea is shown below.

Techniques for the preparation of activated PEG and its conjugation tobiologically active peptides are well known in the art. See, e.g., U.S.Pat. Nos. 5,643,575, 5,919,455, 5,932,462, and 5,990,237, 5,985,265, and5,824,784; Thompson et al., “PEGylation of polypeptides”, EP 0575545 B1;Petit, “Site specific protein modification”, U.S. Pat. Nos. 6,451,986,and 6,548,644; S. Herman et al., “Poly(ethylene glycol) with reactiveendgroups: I. Modification of proteins”, J. Bioactive CompatiblePolymers, 10:145-187, 1995; Y. Lu et al., “PEGylated peptides III:Solid-phase synthesis with PEGylating reagents of varying molecularweight: synthesis of multiply PEGylated peptides”, Reactive Polymers,22:221-229, 1994; A. M. Felix et al., “PEGylated Peptides IV: Enhancedbiological activity of site-directed PEGylated GRF analogs”, Int. J.Peptide Protein Res., 46:253-264, 1995; A. M. Felix, “Site-specificpoly(ethylene glycol)ylation of peptides”, ACS Symposium Series680(poly(ethylene glycol)), 218-238, 1997; Y. Ikeda et al.,“Polyethylene glycol derivatives, their modified peptides, methods forproducing them and use of the modified peptides”, EP 0473084 B1; G. E.Means et al., “Selected techniques for the modification of protein sidechains, in: Chemical modification of proteins”, Holden Day, Inc., pp.219, 1971).

Activated PEG, such as PEG-aldehydes or PEG-aldehyde hydrates, can bechemically synthesized by known means or obtained from commercialsources, e.g., Shearwater Polymers, (Huntsville, Al.) or Enzon, Inc.(Piscataway, N.J.).

An example of a useful activated PEG can be a PEG-aldehyde compound(e.g., a methoxy PEG-aldehyde), such as PEG-propionaldehyde, which iscommercially available from Shearwater Polymers (Huntsville, Al.).PEG-propionaldehyde is represented by the formula PEG-CH₂CH₂CHO. See,e.g., U.S. Pat. No. 5,252,714. Also included within the meaning of “PEGaldehyde compound” are PEG aldehyde hydrates, e.g., PEG acetaldehydehydrate and PEG bis aldehyde hydrate, which latter yields abifunctionally activated structure. See., e.g., Bentley et al.,“Poly(ethylene glycol) aldehyde hydrates and related polymers andapplications in modifying amines”, U.S. Pat. No. 5,990,237. An activatedmulti-branched PEG-aldehyde compound can be used, for example PEGderivatives comprising multiple arms to give divalent, trivalent,tetravalent, octavalent constructs. Using a 4-arm PEG derivative, fourapelin polypeptides can be attached to each PEG molecule. For example,the apelin polypeptide can be conjugated to a polyethylene glycol (PEG)at 1, 2, 3 or 4 amino functionalized sites of the PEG.

In being conjugated, the polyethylene glycol (PEG), as described herein,is covalently bound by alkylation of a thiol present in the peptide oris covalently bound by cycloaddition reaction between azido and alkynemoieties present in the PEG and peptide. Alternatively, the PEG can becovalently bound by reductive amination directly to at least onesolvent-exposed free amine moiety of an amino acid residue of the apelinpolypeptide itself. In some embodiments, the apelin polypeptide can beconjugated to a PEG at one or more primary or secondary amines on thepeptide, or to two PEG groups at a single primary amine site on thepeptide (e.g., this can occur when the reductive amination reactioninvolves the presence of excess PEG-aldehyde compound). It has beenobserved that when PEGylation by reductive amination is at a primaryamine on the peptide, it is not uncommon to have amounts (1 to 100%range) of reaction product that have two or more PEGS present permolecule, and if the desired PEGylation product is one with only one PEGper molecule, then this “over-PEGylation” may be undesirable. WhenPEGylated product with a single PEG per PEGylation product molecule isdesired, an embodiment can be employed that involves PEGylation usingsecondary amines of the pharmacologically active peptide, because onlyone PEG group per molecule will be transferred in the reductiveamination reaction.

Amino acid residues that can provide a primary amine moiety includeresidues of lysine, homolysine, ornithine, α, β-diaminopropionic acid(Dap), α, β-diaminopropionoic acid (Dpr), and α, γ-diaminobutyric acid(Dab), aminobutyric acid (Abu), and α-amino-isobutyric acid (Aib). Thepolypeptide N-terminus also provides a useful α-amino group forPEGylation. Amino acid residues that can provide a secondary aminemoiety include ε-N-alkyl lysine, α-N-alkyl lysine, δ-N-alkyl ornithine,α-N-alkyl ornithine, or an N-terminal proline, where the alkyl is C₁ toC₆.

Another useful activated PEG for generating the PEGylated analogs is aPEG-maleimide compound, such as, but not limited to, a methoxyPEG-maleimide, such as maleimido monomethoxy PEG, are particularlyuseful for generating PEG-conjugated peptides. (e.g., Shen,“N-maleimidyl polymer derivatives”, U.S. Pat. No. 6,602,498; C. Delgadoet al., “The uses and properties of PEG-linked proteins”, Crit. Rev.Therap. Drug Carrier Systems, 9:249-304, 1992; S. Zalipsky et al., “Useof functionalized poly(ethylene glycol)s for modification ofpolypeptides, in: Poly(ethylene glycol) chemistry”, Biotechnical andBiomedical Applications, J. M. Harris, Ed., Plenum Press: New York,347-370, 1992; S. Herman et al., “Poly(ethylene glycol) with reactiveendgroups: I. Modification of proteins”, J. Bioactive CompatiblePolymers, 10:145-187, 1995; P. J. Shadle et al., “Conjugation of polymerto colony stimulating factor-1”, U.S. Pat. No. 4,847,325; G. Shaw etal., “Cysteine added variants IL-3 and chemical modifications thereof”,U.S. Pat. No. 5,166,322 and EP 0469074 B1; G. Shaw et al., “Cysteineadded variants of EPO and chemical modifications thereof”, EP 0668353A1; G. Shaw et al., “Cysteine added variants G-CSF and chemicalmodifications thereof”, EP 0668354 A1; N. V. Katre et al.,“Interleukin-2 muteins and polymer conjugation thereof”, U.S. Pat. No.5,206,344; R. J. Goodson and N. V. Katre, “Site-directed pegylation ofrecombinant interleukin-2 at its glycosylation site”, Biotechnol.,8:343-346, 1990).

A poly(ethylene glycol) vinyl sulfone is another useful activated PEGfor generating the PEG-conjugated apelin polypeptides by conjugation atthiolated amino acid residues, e.g., at C residues. See, e.g., M.Morpurgo et al., “Preparation and characterization of poly(ethyleneglycol) vinyl sulfone”, Bioconj. Chem., 7:363-368, 1996; see alsoHarris, “Functionalization of polyethylene glycol for formation ofactive sulfone-terminated PEG derivatives for binding to proteins andbiologically compatible materials”, U.S. Pat. Nos. 5,446,090; 5,739,208;5,900,461; 6,610,281 and 6,894,025; and Harris, “Water soluble activesulfones of poly(ethylene glycol)”, WO 95/13312 A1. Another activatedform of PEG that is useful in accordance with various embodiments, is aPEG-N-hydroxysuccinimide ester compound, for example, methoxyPEG-N-hydroxysuccinimidyl (NHS) ester. Heterobifunctionally activatedforms of PEG are also useful. See, e.g., Thompson et al., “PEGylationreagents and biologically active compounds formed therewith”, U.S. Pat.No. 6,552,170.

In some embodiments of producing a PEG-apelin peptide conjugate, theapelin polypeptide is reacted by known chemical techniques with anactivated PEG compound, such as but not limited to, a thiol-activatedPEG compound, a diol-activated PEG compound, a PEG-hydrazide compound, aPEG-oxyamine compound, or a PEG-bromoacetyl compound. (See, e.g., S.Herman, “Poly(ethylene glycol) with Reactive Endgroups: I. Modificationof Proteins”, J. Bioactive and Compatible Polymers, 10:145-187, 1995; S.Zalipsky, “Chemistry of Polyethylene Glycol Conjugates with BiologicallyActive Molecules”, Advanced Drug Delivery Reviews, 16:157-182, 1995; R.Greenwald et al., “Poly(ethylene glycol) conjugated drugs and prodrugs:a comprehensive review”, Critical Reviews in Therapeutic Drug CarrierSystems, 17:101-161, 2000).

In another embodiment, the activated PEG for generating a PEG-conjugatedapelin polypeptide can be a multivalent PEG having more than oneactivated residues. Multivalent PEG moieties can include, but are notlimited to, those shown below in Table 8:

TABLE 8 Multivalent PEG

In still other embodiments, the apelin polypeptide can be reacted byknown chemical techniques with an activated multi-branched PEG compound(PEG derivatives comprising multiple arms to give divalent, trivalent,tetravalent, octavalent constructs), such as but not limited to,pentaerythritol tetra-polyethyleneglycol ether. Functionalization andactivated derivatives, such as, but not limited to,N-succinimidyloxycarbonyl)propyl, p-nitrophenyloxycarbonyl,(—CO₂-p-C₆H₄NO₂), 3-(N-maleimido)propanamido, 2-sulfanylethyl, and3-aminopropyl. Using a 4-arm PEG derivative, four apelin polypeptidescan be attached to each PEG molecule. For example, the apelinpolypeptide can be conjugated to a polyethylene glycol (PEG) at:

1, 2, 3 or 4 amino functionalized sites of the PEG;

1, 2, 3 or 4 thiol functionalized sites of the PEG;

1, 2, 3 or 4 maleimido functionalized sites of the PEG;

1, 2, 3 or 4 N-succinimidyl functionalized sites of the PEG;

1, 2, 3 or 4 carboxyl functionalized sites of the PEG;

1, 2, 3 or 4 p-nitrophenyloxycarbonyl functionalized sites of the PEG;

1, 2, 3 or 4 azido functionalized sites of the PEG;

1, 2, 3 or 4 alkene or alkyne functionalized sites of the PEG; or

1, 2, 3 or 4 halide or halide-acetylamide functionalized sites of thePEG.

The smallest practical size of PEG is about 500 Daltons (Da), belowwhich PEG becomes toxic. Above about 500 Da, any molecular mass for aPEG can be used as practically desired, e.g., from about 500 Daltons(Da) to 100,000 Da (n is 10 to 2300). The number of PEG monomers (n) isapproximated from the average molecular mass using a MW=44 Da for eachmonomer. In some embodiments, the combined or total average molecularmass of PEG used in a PEG-conjugated apelin polypeptide can be fromabout 500 Da to 10,000 Da (total n is from 10 to 230), or from about10,000 to 40,000 Da (total n is from 230 to 910), or from about 40,000to 100,000 Da (total n is from 910 to 2300).

In various other embodiments, the combined molecular mass of the PEGmolecule should not exceed about 100,000 Da. In some embodiments, thecombined or total average molecular mass of PEG used in a PEG-conjugatedapelin polypeptide can be from about 3,000 Da to 60,000 Da (total n isfrom 70 to 1,400), from about 10,000 Da to 40,000 Da (total n is about230 to about 910). In other embodiments the combined mass for PEG isfrom about 20,000 Da to 30,000 Da (total n is about 450 to about 680).In one embodiment, the average molecular mass of PEG used in aPEG-conjugated apelin polypeptide is about 5,000 Da (5 kDa). In anotherembodiment, the average molecular mass of PEG used in a PEG-conjugatedapelin polypeptide is about 10,000 Da (10 kDa). In still anotherembodiment, the average molecular mass of PEG used in a PEG-conjugatedapelin polypeptide is about 20,000 Da (20 kDa).

In some embodiments, the PEG polymer is conjugated to the apelinpolypeptide through a conjugation linker as described in detail below.In one embodiment, the conjugation linker comprises 3-mercaptopropanoicacid (abbreviated as MerPr herein). In another embodiment, theconjugation linker comprises 3-(1H-1,2,3-triazol-4-yl)propanoic acid(abbreviated as 3TP herein). 3TP results from pent-4-ynoic acid when aPEG azide click reaction is employed for the conjugation. ExemplaryPEG-apelin peptide conjugates of the invention are shown in Table 9below. PEG abbreviations used in Table 9 and elsewhere herein aredefined as follows:NPeg11=1-amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oicacid;[Pra](NPeg9)=(S)-2-amino-3-(1-(32-amino-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)-1H-1,2,3-triazol-4-yl)propanoicacid; 10K-mPEGacetamideReg, 10K-mPEGReg, or10K-mPEGacetamide=Methoxypolyethylene glycol amine-N-acetamide (10 kDa);and 20K-mPEGacetamideReg, 20K-mPEGReg,20K-mPEGacetamide=Methoxypolyethylene glycol amine-N-acetamide (20 kDa).Note that MerPr and 3TP, which are both linkers as defined above, arepositioned between the PEG polymer and the amino terminus of the apelinpolypeptide even though the linker abbreviation is listed first in thesequences below. In certain embodiments, the modified apelin polypeptidemay comprise an amino acid sequence of any one of the peptides listed inTable 9 below.

TABLE 9 Exemplary PEG-Apelin Peptide Conjugates SEQ ID NO: SEQUENCE 96{H2}[PE]RP[hArg][Cha][Pra](NPeg9)HKG [Oic][Nle]P[4-Cl-F]{COOH} 97[MerPr](20K mPEGacetamide)KFRRQRP[hArg][Cha]SHKG[Oic][Nle]P[4-Cl-F]{COOH} 98 {H2}[MerPr](20K mPEGacetamide)FRRQRP[hArg][Cha]SHKG[Oic][Nle] P[4-Cl-F]{COOH} 99 {H2}[MerPr](20KmPEGacetamide)RRQRP[hArg] [Cha]SHKG[Oic][Nle]P[4-Cl-F]{COOH} 100{H2}[MerPr](20K mPEGacetamide)RQRP[hArg][Cha]SHKG[Oic][Nle]P[4-Cl-F]{COOH} 101 {H2}[MerPr](20KmPEGacetamide)QRP[hArg][Cha]SHKG [Oic][Nle]P[4-Cl-F]{COOH} 102{Npeg11}KFRRQRP[hArg][Cha]SHKG[Oic][Nle] P[4-Cl-F]{COOH} 103{H2}[3TP](20K-mPEGReg)[hArg]rQ[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 104{H2}[3TP](20K-mPEGReg)[hArg]RQ[hArg]PR[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 105{Acetyl}[Atz(20K-mPEGReg)]KFRRQRP[hArg][Cha]SHKG[Oic][Nle]P[4-Cl-F]{COOH} 106{Acetyl}[Atz(20K-mPEGReg)]LRP[hArg][Cha]SHKG [Oic][Nle]P[4-Cl-F]{COOH}107 {Acetyl-NH}[Pra](20K-mPEGReg)OF[hArg] [hArg]QRP[hArg][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 108{Acetyl-NH}[Pra](20K-mPEGReg)OF[hArg][hArg] QRP[hArg][NMeLeu]SHKG[Oic][pI-Phe]P[pI-Phe]{COOH} 109{Acetyl-NH}[NPeg11]QRP[hArg][Cha]SHKG [Oic][Nle]P[4-Cl-F]{COOH} 110{H2}[MerPr](10K mPEGacetamide)QRPRLSHKGPMPF {COOH} 111{H2}[MerPr](10KmPEGacetamide)q[hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[D-4ClF]{COOH} 112{H2}[MerPr](10KmPEGacetamide)Q[hArg]P[NMeArg][aMeLeu]SHKGP[Nle]P[4-Cl-F]{COOH} 113{H2}[MerPr](10KmPEGacetamide)Q[hArg]P[NMehArg]LSHKGP[Nle]P[4-Cl-F]{COOH} 114{H2}[MerPr](10KmPEGacetamide)QR[aMePro][hArg][aMeLeu]SHKGP[Nle]P[4-Cl-F]{COOH} 115{H2}[MerPr](10KmPEGacetamide)QRP [NMeArg]LSHKGP[Nle]P[4-Cl-F]{COOH} 116{H2}[MerPr](10KmPEGacetamide)Q[NMehArg]P[NMeArg][aMeLeu]SHKGP[Nle]P[4-Cl-F]{COOH} 117{H2}[MerPr](10KmPEGacetamide)QRP[hArg] [aMeLeu]SHKGP[Nle]P[4-Cl-F]{COOH}119 {H2}[MerPr](10KmPEGacetamide)QrP[hArg][aMeLeu]SHKGP[Nle]P[4-Cl-F]{COOH} 120{H2}[MerPr](10KmPEGacetamide)QRP[NMeArg][Cha]SHKG[Oic][Nle][Aib][4-Cl-F]{COOH}

In certain embodiments of the invention, the half-life extension moietyor vehicle is an immunoglobulin or immunoglobulin Fc domain,particularly a human immunoglobulin or human immunoglobulin Fc domain(e.g. IgG1, IgG2, IgG3 or IgG4). Thus, any of the modified apelinpolypeptides disclosed herein can be conjugated, optionally through aconjugation linker, to an immunoglobulin or immunoglobulin Fc domain. Inembodiments in which the conjugation linker is present, the conjugationlinker may be a peptidyl or non-peptidyl linker. In certain embodiments,the non-peptidyl linker comprises a PEG polymer. The term“immunoglobulin” encompasses full antibodies comprising two dimerizedheavy chains (HC), each covalently linked to a light chain (LC); asingle undimerized immunoglobulin heavy chain and covalently linkedlight chain (HC+LC); or a chimeric immunoglobulin (light chain+heavychain)-Fc heterotrimer (a so-called “hemibody”). An “Fc region”, or usedinterchangeably herein, “Fc domain” or “immunoglobulin Fc domain,”contains two heavy chain fragments, which can comprise the CH1, CH2, andCH3 domains of a HC of an antibody. In some embodiments, the Fc domaincomprises the immunoglobulin CH2 and CH3 domains, but not the CH1domain. The two heavy chain fragments of an Fc domain are held togetherby two or more disulfide bonds and by hydrophobic interactions of theCH3 domains.

Recombinant fusion or chemical conjugation of the apelin polypeptides ofthe invention to a recombinant immunoglobulin or immunoglobulin Fcdomain of any of the IgG1, IgG2, IgG3 or IgG4 isotypes can be useful toextend pharmacokinetic half-life. See, e.g., WO 2010/108153A2. Any ofthe carrier immunoglobulins or Fc domains thereof disclosed in WO2010/108153 A2 or WO/2012/040518, or isotype conversions of any of themcomprising different isotype constant domains, or other carrierimmunoglobulins known in the art, can be used as half-life extendingmoieties within the scope of the invention. For example, aglycosylated(e.g., N297G variant IgG1; WO 2012/075037 A1) and/orcysteine-substituted (“CysMab”) variant immunoglobulin Fc monomers canalso be employed for enhanced stability or modified effector function.See, e.g., WO2007/022070 A2, which is hereby incorporated by referencein its entirety.

One example of a human IgG2 heavy chain (HC) constant domain that can beused to produce an immunoglobulin or Fc domain half-life extensionmoiety has the amino acid sequence:

(SEQ. ID NO: 729) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Constant region sequences of other IgG isotypes are known in the art foran IgG1, IgG2, IgG3, or IgG4 immunoglobulin isotype, if desired. Ingeneral, human IgG2 can be used for targets where effector functions arenot desired, and human IgG1 in situations where such effector functions(e.g., antibody-dependent cytotoxicity (ADCC)) are desired. Human IgG3has a relatively short half-life and human IgG4 forms antibody“half-molecules.” There are four known allotypes of human IgG1. Thepreferred allotype is referred to as “hIgG1z”, also known as the “KEEM”allotype. Human IgG1 allotypes “hIgG1za” (KDEL), “hIgG1f” (REEM), and“hIgG1fa” are also useful; all appear to have ADCC effector function.

Human hIgG1z heavy chain (HC) constant domain has the amino acidsequence:

(SEQ ID NO: 730) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Human hIgG1za heavy chain (HC) constant domain has the amino acidsequence:

(SEQ ID NO: 731) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Human hIgG1f heavy chain (HC) constant domain has the amino acidsequence:

(SEQ ID NO: 732) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Human hIgG1fa heavy chain (HC) constant domain has the amino acidsequence:

(SEQ ID NO: 733) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In some embodiments of the invention, the half-life extending moiety isan immunoglobulin Fc domain (e.g., a human immunoglobulin Fc domain,including Fc of allotype IgG1, IgG2, IgG3 or IgG4) or a portion thereof(e.g., CH2 domain of the Fc domain). The Fc domain may comprise thesequence of any one of SEQ ID NOs: 716 and 729-733. In one embodiment,the Fc domain to which an apelin polypeptide of the invention isconjugated comprises the amino acid sequence of SEQ ID NO: 716.

Monovalent dimeric or bivalent dimeric Fc-apelin peptide fusions orconjugates are useful embodiments of the peptide conjugates of theinvention. A “monovalent dimeric” Fc-apelin peptide fusion or conjugate,or interchangeably, “monovalent dimer,” or interchangeably, “monovalentheterodimer,” is an Fc-apelin peptide analog fusion or conjugate thatincludes an apelin peptide conjugated with only one of the dimerized Fcdomains. A “bivalent dimeric” Fc-apelin peptide analog fusion, orinterchangeably, “bivalent dimer” or “bivalent homodimer,” is aFc-apelin peptide fusion or conjugate having both of the dimerized Fcdomains each conjugated separately with an apelin peptide analog.

Immunoglobulin Fc domains include Fc variants, which are suitablehalf-life extending moieties within the scope of this invention. Anative Fc can be extensively modified to form an Fc variant inaccordance with this invention, provided binding to the salvage receptoris maintained; see, for example WO 97/34631, WO 96/32478, and WO04/110472. In such Fc variants, one can remove one or more sites of anative Fc that provide structural features or functional activity notrequired by the fusion or conjugate molecules of this invention. One canremove these sites by, for example, substituting or deleting residues,inserting residues into the site, or truncating portions containing thesite. The inserted or substituted residues can also be altered aminoacids, such as peptidomimetics or D-amino acids. Fc variants can bedesirable for a number of reasons, several of which are described below.Exemplary Fc variants include molecules and sequences in which:

1. Sites involved in disulfide bond formation are removed. Such removalcan avoid reaction with other cysteine-containing proteins present inthe host cell used to produce the molecules of the invention. For thispurpose, the cysteine-containing segment at the N-terminus can betruncated or cysteine residues can be deleted or substituted with otheramino acids (e.g., alanyl, seryl). Even when cysteine residues areremoved, the single chain Fc domains can still form a dimeric Fc domainthat is held together non-covalently.

2. A native Fc is modified to make it more compatible with a selectedhost cell. For example, one can remove the PA dipeptide sequence nearthe N-terminus of a typical native Fc, which can be recognized by adigestive enzyme in E. coli such as proline iminopeptidase. One can alsoadd an N-terminal methionine residue, especially when the molecule isexpressed recombinantly in a bacterial cell such as E. coli.

3. A portion of the N-terminus of a native Fc is removed to preventN-terminal heterogeneity when expressed in a selected host cell. Forthis purpose, one can delete any of the first 20 amino acid residues atthe N-terminus, particularly those at positions 1, 2, 3, 4 and 5.

4. One or more glycosylation sites are removed. Residues that aretypically glycosylated (e.g., asparagine) can confer cytolytic response.Such residues can be deleted or substituted with unglycosylated residues(e.g., alanine).

5. Sites involved in interaction with complement, such as the C1qbinding site, are removed. For example, one can delete or substitute theEKK tripeptide sequence of human IgG1. Complement recruitment may not beadvantageous for the molecules of this invention and so can be avoidedwith such an Fc variant.

6. Sites are removed that affect binding to Fc receptors other than asalvage receptor. A native Fc can have sites for interaction withcertain white blood cells that are not required for the fusion orconjugate molecules of the present invention and so can be removed.

7. The ADCC site is removed to decrease or eliminate ADCC effectorfunction, or alternatively, modified for enhanced ADCC effector functionby non-fucosylation or de-fucosylation. ADCC sites are known in the art;see, for example, Molec. Immunol. 29(5): 633-9, 1992, with regard toADCC sites in IgG1. These sites, as well, are not required for thefusion or conjugate molecules of the present invention and so can beremoved, or enhanced for ADCC effector function, as may be desired. See,Iida et al., Two mechanisms of the enhanced antibody-dependent cellularcytotoxicity (ADCC) efficacy of non-fucosylated therapeutic antibodiesin human blood, BMC Cancer 9:58, 2009, doi:10.1186/1471-2407-9-58.

8. When the native Fc is derived from a non-human antibody, the nativeFc can be humanized. Typically, to humanize a native Fc, one willsubstitute selected residues in the non-human native Fc with residuesthat are normally found in human native Fc. Techniques for antibodyhumanization are well known in the art.

9. A peptidyl or non-peptidyl linker of suitable length and neutralcharge, such as those described in more detail below can be covalentlyfused between N-terminus of the apelin polypeptide of the invention andthe C-terminus, N-terminus, or an internal residue of an Fc domain. Insome embodiments, the Fc domain can be engineered to include an internalresidue (such as a cysteine residue) to which the apelin polypeptide maybe conjugated (see Example 11). Other engineered Fc domains that can beused in the Fc-apelin peptide conjugates are described in WO2007/022070A2, which is hereby incorporated by reference in its entirety. Forpurposes of the invention, a variant Fc domain can also be part of amonomeric immunoglobulin heavy chain, an antibody, or a heterotrimerichemibody (LC+HC+Fc).

It will be appreciated that “multimers” of the peptide conjugates can bemade, since the half-life extending moiety employed for conjugation ofthe apelin polypeptide (with or without an intervening linker moiety)can be multivalent (e.g., bivalent, trivalent, tetravalent or a higherorder valency) as to the number of amino acid residues at which thehalf-life extending moiety can be conjugated. In some embodiments, thepeptide can be multivalent (e.g., bivalent, trivalent, tetravalent or ahigher order valency), and, thus, some “multimers” may have more thanone half-life extending moiety. Consequently, it is possible to producea variety of conjugated half-life extending moiety peptide structures.By way of example, a univalent half-life extending moiety and aunivalent peptide can produce a 1:1 conjugate; a bivalent peptide and aunivalent half-life extending moiety may form conjugates wherein thepeptide conjugates bear two half-life extending moiety moieties, whereasa bivalent half-life extending moiety and a univalent peptide mayproduce species where two peptide entities are linked to a singlehalf-life extending moiety; use of higher-valence half-life extendingmoiety can lead to the formation of clusters of peptide entities boundto a single half-life extending moiety, whereas higher-valence peptidesmay become encrusted with a plurality of half-life extending moietymoieties. By way of further example, if the site of conjugation of amultivalent half-life extending moiety to the apelin polypeptide is acysteine or other aminothiol, the methods disclosed by D'Amico et al.may be employed (D'Amico et al., “Method of conjugating aminothiolcontaining molecules to vehicles”, published as U.S. Patent PublicationNo. 2006/0199812, which application is incorporated herein by referencein its entirety).

The peptide moieties may have more than one reactive group which willreact with the activated half-life extending moiety and the possibilityof forming complex structures must always be considered; when it isdesired to form simple structures such as 1:1 adducts of half-lifeextending moiety and peptide, or to use bivalent half-life extendingmoiety to form peptide:half-life extending moiety:peptide adducts, itwill be beneficial to use predetermined ratios of activated half-lifeextending moiety and peptide material, predetermined concentrationsthereof and to conduct the reaction under predetermined conditions (suchas duration, temperature, pH, etc.) so as to form a proportion of thedescribed product and then to separate the described product from theother reaction products. The reaction conditions, proportions andconcentrations of the reagents can be obtained by relatively simpletrial-and-error experiments which are within the ability of anordinarily skilled artisan with appropriate scaling-up as necessary.Purification and separation of the products is similarly achieved byconventional techniques well known to those skilled in the art.

Additionally, physiologically acceptable salts of the half-lifeextending moiety-fused or conjugated to the apelin polypeptide are alsoencompassed by various embodiments described within.

The above-described half-life extending moieties and other half-lifeextending moieties described herein are useful, either individually orin combination, and as further described in the art, for example, inU.S. Patent Publication No. 2007/0071764 and WO 2008/088422, which areboth incorporated herein by reference in their entireties. Variousembodiments encompass the use of any single species of pharmaceuticallyacceptable half-life extending moiety, such as, but not limited to,those described herein, in conjugation with the apelin polypeptides, orthe use of a combination of two or more like or different half-lifeextending moieties.

The modified apelin polypeptides of the invention may, in someembodiments, be conjugated or attached to a half-life extension moietythrough a conjugation linker. The linker, e.g., may consist of athioether, amine, imine, amide, triazole, disulfide, or a carbon-carbonbond. Thus, in various embodiments, e.g. a thioether can tether thevehicle or half-life extension moiety to the apelin polypeptide. Incertain embodiments, the conjugation linker, when present, comprisesAeea, Aeea-Aeea, γGlu-Aeea, γGlu-Aeea-Aeea, or γGlu. See, e.g. Example 2herein.

The conjugation linker or linker moiety can be a biologically acceptablepeptidyl or non-peptidyl organic group that is covalently bound to anamino acid residue of an apelin polypeptide or other polypeptide chain(e.g., an immunoglobulin heavy chain or light chain or immunoglobulin Fcdomain) contained in a composition, which linker moiety covalently joinsor conjugates the apelin polypeptide or other polypeptide chain toanother peptide or polypeptide chain in the composition, or to ahalf-life extending moiety. In some embodiments, a half-life extendingmoiety, as described herein, is conjugated, i.e., covalently bounddirectly to an amino acid residue of the apelin polypeptide itself, oroptionally, to a peptidyl or non-peptidyl linker moiety (including butnot limited to aromatic or aryl linkers) that is covalently bound to anamino acid residue of the apelin polypeptide.

In some embodiments, the presence of a linker moiety can be useful inoptimizing pharmacological activity of the peptide conjugates. Thelinker may be made up of amino acids linked together by peptide bonds.The linker moiety, if present, can be independently the same ordifferent from any other linker, or linkers, that may be present in thecomposition. As stated above, the linker moiety, if present (whetherwithin the primary amino acid sequence of the apelin peptide, or as alinker for attaching a half-life extending moiety to the apelinpeptide), can be “peptidyl” in nature (i.e., made up of amino acidslinked together by peptide bonds) and made up in length, preferably, offrom 1 up to about 40 amino acid residues, of from 1 up to about 20amino acid residues, or from 1 to about 10 amino acid residues. Theamino acid residues in the linker are from among the twenty canonicalamino acids, e.g., cysteine, glycine, alanine, proline, asparagine,glutamine, and/or serine. In various embodiments, a peptidyl linker canbe made up of a majority of amino acids that are sterically unhindered,such as glycine, serine, and alanine linked by a peptide bond. It mayalso be desirable that, if present, a peptidyl linker be selected thatavoids rapid proteolytic turnover in circulation in vivo. Some of theseamino acids may be glycosylated, as is well understood by those in theart. For example, a useful linker sequence constituting a sialylationsite is X₁X₂NX₄X₅G (SEQ ID NO: 734), wherein X₁, X₂, X₄ and X₅ are eachindependently any amino acid residue. It may also be desirable that, ifpresent, a peptidyl linker can consist of any non-canonical amino acidsor a combination of non-canonical and canonical amino acids selected toavoid or reduce rapid proteolytic turnover in vitro and/or in vivo.

In other embodiments, the 1 to 40 amino acids of the peptidyl linkermoiety can be selected from glycine, alanine, proline, asparagine,glutamine, and lysine. A linker can be made up of a majority of aminoacids that are sterically unhindered, such as glycine and alanine. Thus,linkers can include polyglycines, polyserines, and polyalanines, orcombinations of any of these. Some exemplary peptidyl linkers arepoly(Gly)₁₋₈, particularly (Gly)₃ (SEQ ID NO: 735), (Gly)₄ (SEQ ID NO:736), (Gly)₅ (SEQ ID NO: 737) and (Gly)₇ (SEQ ID NO: 738), as well as,poly glycine-serine linkers, such as “L15” (GGGGSGGGGSGGGGS; SEQ ID NO:739), poly glycine-alanine and polyalanine linkers. Other specificexamples of peptidyl linkers include (Gly)₅Lys (SEQ ID NO: 740), and(Gly)₅LysArg (SEQ ID NO: 741). Other examples of useful peptidyl linkersare:

(Gly)₃Lys(Gly)₄ (SEQ ID NO: 742);

(Gly)₃AsnGlySer(Gly)₂ (SEQ ID NO: 743);

(Gly)₃Cys(Gly)₄ (SEQ ID NO: 744); and

GlyProAsnGlyGly (SEQ ID NO: 745).

To explain the above nomenclature, for example, (Gly)₃Lys(Gly)₄ (SEQ IDNO: 742) means Gly-Gly-Gly-Lys-Gly-Gly-Gly-Gly (SEQ ID NO:742). Othercombinations of Gly and Lys or Gly and Ala are also useful.

Other linkers are those identified herein as “L5” (GGGGS; or “G₄S”; SEQID NO: 746), “L10” (GGGGSGGGGS; SEQ ID NO: 747); “L20”(GGGGSGGGGSGGGGSGGGGS; SEQ ID NO: 748); “L25”(GGGGSGGGGSGGGGSGGGGSGGGGS; SEQ ID NO: 749) and any linkers used in theworking examples hereinafter.

In some embodiments which comprise a peptide linker moiety, acidicresidues, for example, glutamate or aspartate residues are placed in theamino acid sequence of the linker moiety. Examples include the followingpeptide linker sequences:

(SEQ ID NO: 750) GGEGGG; (SEQ ID NO: 751) GGEEEGGG; (SEQ ID NO: 752)GEEEG; (SEQ ID NO: 753) GEEE; (SEQ ID NO: 754) GGDGGG; (SEQ ID NO: 755)GGDDDGG; (SEQ ID NO: 756) GDDDG; (SEQ ID NO: 757) GDDD; (SEQ ID NO: 758)GGGGSDDSDEGSDGEDGGGGS; (SEQ ID NO: 759) WEWEW; (SEQ ID NO: 760) FEFEF;(SEQ ID NO: 761) EEEWWW; (SEQ ID NO: 762); EEEFFF; (SEQ ID NO: 763)WWEEEWW; or (SEQ ID NO: 764) FFEEEFF.

The linkers shown here are exemplary; peptidyl linkers may be muchlonger and may include other residues. A peptidyl linker can contain,e.g., a cysteine, another thiol, or nucleophile for conjugation with ahalf-life extending moiety. In another embodiment, the linker cancontain a cysteine or homocysteine residue, or other 2-amino-ethanethiolor 3-amino-propanethiol moiety for conjugation to maleimide,iodoacetamide or thioester, functionalized half-life extending moiety.

Another useful peptidyl linker is a large, flexible linker comprising arandom Gly/Ser/Thr sequence, for example: GSGSATGGSGSTASSGSGSATH (SEQ IDNO: 765) or HGSGSATGGSGSTASSGSGSAT (SEQ ID NO: 766), which is estimatedto be about the size of a 1 kDa PEG molecule. Alternatively, a usefulpeptidyl linker may be comprised of amino acid sequences known in theart to form rigid helical structures (e.g., Rigid linker:-AEAAAKEAAAKEAAAKAGG (SEQ ID NO: 767). Additionally, a peptidyl linkercan also comprise a non-peptidyl segment such as a 6 carbon aliphaticmolecule of the formula —CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—. The peptidyl linkerscan be altered to form derivatives as described herein.

A non-peptidyl linker moiety is also useful for conjugating thehalf-life extending moiety to the apelin peptide. For example, alkyllinkers such as —NH—(CH₂)_(s)—C(O)—, wherein s=2-20 can be used. Thesealkyl linkers may further be substituted by any non-sterically hinderinggroup such as lower alkyl (e.g., C₁-C₆) lower acyl, halogen (e.g., Cl,Br), CN, NH₂, phenyl, etc. Exemplary non-peptidyl linkers are PEGlinkers (e.g., shown below):

wherein n is such that the linker has a molecular weight of about 100 toabout 5000 Daltons (Da). In certain embodiments, the non-peptidyl linkeris bromoacetyl-NPeg11 as illustrated in Example 11 for an Fc-apelinpeptide conjugate.

The linker could also be an unnatural amino acid, such as aminohenxanoicacid, or an organic group, such as a dicarbohylic acid, such as succinicacid (butanedioic acid).

In one embodiment, the non-peptidyl linker is aryl. The linkers may bealtered to form derivatives in the same manner as described herein. Inaddition, PEG moieties may be attached to the N-terminal amine orselected side chain amines by either reductive alkylation using PEGaldehydes or acylation using hydroxysuccinimido or carbonate esters ofPEG, or by thiol conjugation.

“Aryl” is phenyl or phenyl vicinally-fused with a saturated,partially-saturated, or unsaturated 3-, 4-, or 5 membered carbon bridge,the phenyl or bridge being substituted by 0, 1, 2 or 3 substituentsselected from C₁₋₈ alkyl, C₁₋₄ haloalkyl or halo. “Heteroaryl” is anunsaturated 5, 6 or 7 membered monocyclic or partially-saturated orunsaturated 6-, 7-, 8-, 9-, 10- or 11 membered bicyclic ring, wherein atleast one ring is unsaturated, the monocyclic and the bicyclic ringscontaining 1, 2, 3 or 4 atoms selected from N, O and S, wherein the ringis substituted by 0, 1, 2 or 3 substituents selected from C₁₋₈ alkyl,C₁₋₄ haloalkyl and halo. Non-peptide portions, such as non-peptidyllinkers or non-peptide half-life extending moieties can be synthesizedby conventional organic chemistry reactions.

The above is merely illustrative and not an exhaustive treatment of thekinds of linkers that can optionally be employed in accordance withvarious embodiments of the half-life extension-apelin peptide conjugatesof the invention.

As an additional strategy to increase the stability of apelinpolypeptides, polypeptides can be prepared in which a covalent bondjoins any two atoms present in the polypeptide to form a cyclicstructure. X_(n) and X_(n+3) or X_(n) and X_(n+4) are independently anatural or unnatural amino acid selected from K, D, Orn, Dab or E ineither D- or L-stereochemistry where the side chains of the amino acidsat these positions are covalently linked together forming an amide bond(—NHC(O)— or —C(O)—NH—). The amide bond may be formed directly via theside chain amino and carboxyl functional groups or with a diamino,bis-carboxyl, or amino-carboxyl linker forming the amide bonds.Alternatively, the amide bond can be replaced with either a monosulfide(—S—), a disulfide (—S—S—) or a linkage of Formula —S—CH2-C(═Z)—CH2-S—;wherein Z is O, N—O—CH2C(O)— to accomplish cyclization. In someembodiments, the cyclic apelin polypeptides are conjugated to ahalf-life extension moiety to further enhance the stability of thepeptides and/or modulate the pharmacokinetic profile of the peptides.

In one embodiment, a modified apelin polypeptide of the invention can bedescribed by the following formula:N₁[AC4Abu]N₂X,wherein: N₁ is a fatty acyl group or Acetyl-NH, N₂ is [Aeea][Aeea] or[Aeea], and X is an apelin polypeptide of 11, 12, 13, 14, 15, 16 or 17amino acids long comprising E1xxK1 (cyclized via an amide bond between Eand K) wherein xx is 2-3 amino acids selected from D- or L-amino acids,α- or β-amino acids, homologated amino acids, N-methylated amino acids,α-methyl amino acids, amino acids bearing the side chain on N instead ofthe α-carbon or another canonical or a non-canonical amino acid.

In another embodiment, a modified apelin polypeptide of the inventioncan be described by the following formula:[Z]X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂ X₁₃PX₁₄  (SEQ ID NO: 768)having only one amide bond between the carboxylate side chain of E andthe amino sidechain of K and wherein: Z is acetyl, acyl, [Atz(PEG10)], aPEG derivative, {TDA}[AC4Abu][Aeea][Aeea], or other half-life extensionmoiety or is absent; X₁ is r, [hArg], [NmeArg], R, absent, or E and ifX₁ is E its carboxylate sidechain forms an amide bond with the sidechainof a K at position X₄ or X₈; X₂ is r, [hArg], [NmeArg], R, absent, or Eand if X₂ is E its carboxylate sidechain forms an amide bond with thesidechain of a K at position X₈ or X₆; X₃ is Q, q, or E and if X₃ is Eits carboxylate sidechain forms an amide bond with the sidechain of a Kat position X₆ or X₇; X₄ is r, [hArg], [NmeArg], R, K or E and if X₄ isE its carboxylate sidechain forms an amide bond with the sidechain of aK at position X₇ or X₈, or if X₄ is K its amine sidechain forms an amidebond with the sidechain of E at position X₁; X₅ is P, K or E and if X₅is E its carboxylate sidechain forms an amide bond with the sidechain ofa K at position X₈ or X₉, or if X₅ is K its amine sidechain forms anamide bond with the sidechain of E at position X₁ or X₂; X₆ is r,[hArg], [NmeArg], R, K or E and if X₆ is E its carboxylate sidechainforms an amide bond with the sidechain of a K at position X₉ or X₁₀, orif X₆ is K its amine sidechain forms an amide bond with the sidechain ofE at position X₂ or X₃; X₇ is [Cha], [NmeLeu], K or E and if X₇ is E itscarboxylate sidechain forms an amide bond with the sidechain of a K atposition X₁₀ or X₁₁, or if K is X₇ its amine sidechain forms an amidebond with the sidechain of E at position X₃ or X₄; X₈ is S, K or E andif X₈ is E its carboxylate sidechain forms an amide bond with thesidechain of a K at position X₁₀ or X₁₁, or if X₈ is K its aminesidechain forms an amide bond with the sidechain of E at position X₄ orX₈; X₉ is H, or K and if X₉ is E its amine sidechain forms an amide bondwith the sidechain of E at position X₈ or X₆; X₁₀ is K and its aminesidechain can, but does not necessarily form an amide bond with thesidechain of E at position X₆ or X₇; X₁₁ is G, or K and if X₁₁ is K itsamine sidechain forms an amide bond with the sidechain of E at positionX₇ or X₈; X₁₂ is [Oic], or K and if X₁₂ is K its amine sidechain formsan amide bond with the sidechain of E at position X₈ or X₉; X₁₃ is[p-I-Phe], [Nle], or K and if X₁₃ is E its amine sidechain forms anamide bond with the sidechain of E at position X₉, and X₁₄ is [D-Bip],[D-4ClF] or [4-Cl-F].

Exemplary cyclic apelin polypeptides conjugated to a lipid moiety areshown in Table 10. E1xxK1 or E1xxxK1 (underlined) indicates the positionof cyclization via the side chains of the E and K residues.

TABLE 10 Exemplary Cyclic Apelin Polypeptides SEQ ID NO: SEQUENCE 648{TDA}[AC4Abu][Aeea][Aeea]Q[hArg]Pr[Cha]E1HKK1 [Oic][Nle]P[4-Cl-F]{COOH}649 {TDA}[AC4Abu][Aeea][Aeea]q[hArg]Pr[Cha]E1HKK1[Oic][Nle]P[4-Cl-F]{COOH} 650 {TDA}[AC4Abu][Aeea][Aeea]QrPr[Cha]E1HKK1[Oic][Nle]P[4-Cl-F]{COOH} 651{TDA}[AC4Abu][Aeea][Aeea]q[hArg]PE1[Cha]SK1KG [Oic][Nle]P[4-Cl-F]{COOH}652 {TDA}[AC4Abu][Aeea][Aeea]QrPE1[Cha]SK1KG[Oic] [Nle]P[4-Cl-F]{COOH}653 {TDA}[AC4Abu][Aeea][Aeea]rQ[hArg]PE1[Cha]SK1KG[Oic][Nle]P[4-Cl-F]{COOH} 654{TDA}[AC4Abu][Aeea][Aeea]E1[hArg]QK1Pr[Cha]SHKG[Oic][Nle]P[4-Cl-F]{COOH} 655{TDA}[AC4Abu][Aeea][Aeea]QE1Pr[Cha]K1HKG [Oic][Nle]P[4-Cl-F]{COOH} 656{TDA}[AC4Abu][Aeea][Aeea]rQE1Pr[Cha]K1HKG [Oic][Nle]P[4-Cl-F]{COOH} 657{TDA}[AC4Abu][Aeea][Aeea]E1Q[hArg]PK1[Cha] SHKG[Oic][Nle]P[4-Cl-F]{COOH}658 {TDA}[AC4Abu][Aeea][Aeea]E1q[hArg]PK1[Cha]SHKG[Oic][Nle]P[4-Cl-F]{COOH} 659 {TDA}[AC4Abu][Aeea][Aeea]E1QrPK1[Cha]SHKG[Oic][Nle]P[4-Cl-F]{COOH} 660{TDA}[AC4Abu][Aeea][Aeea]E1[hArg]Q[hArg]K1r[Cha]SHKG[Oic][Nle]P[4-Cl-F]{COOH} 661{Acetyl-NH}r[hArg]Q[hArg]PE1[NMeLeu]SK1KG [Oic][pI-Phe]P[D-Bip]{COOH}662 {Acetyl-NH}[hArg][hArg]q[hArg]PE1[NMeLeu]SK1KG[Oic][pI-Phe]P[D-Bip]{COOH} 663{Acetyl-NH}[hArg][hArg]QrPE1[NMeLeu]SK1KG [Oic][pI-Phe]P[D-Bip]{COOH}664 {Acetyl-NH}r[hArg]Q[hArg]PE1[Cha]SK1KG[Oic] [pI-Phe]P[D-Bip]{COOH}665 {Acetyl-NH}[hArg]rQ[hArg]PE1[Cha]SK1KG[Oic] [pI-Phe]P[D-Bip]{COOH}666 {Acetyl-NH}[hArg][hArg]q[hArg]PE1[Cha]SK1KG[Oic][pI-Phe]P[D-Bip]{COOH} 667 {Acetyl-NH}[hArg][hArg]QrPE1[Cha]SK1KG[Oic][pI-Phe]P[D-Bip]{COOH}

In some embodiments, a modified apelin polypeptide of the invention canbe described by the following formula:

(SEQ ID NO: 769) Z₁ Z₂ X₁ X₂ X₃ X₄X₅ X₆X₇ X₈X₉ X₁₀X₁₁X₁₂ X₁₃ X₁₄X₁₅ X₁₆ X₁₇,Wherein Z₁ is an acyl group; Z₂ comprises a conjugation linker or isabsent; X₁ is O, K, [D-Orn], [k], [BLys], [D-BLys], [BhLys], or[D-BhLys] or is absent; X₂ is F, [BhPhe], [BPhe] or is absent; X₃ is R,[hArg], [r], [NMeArg], [NMehArg], [rhArg], [rArg], [BhArg] or is absent;X₄ is R, [hArg], [r], [NMeArg], [NMehArg], [rhArg], [rArg], [BhArg] oris absent; X₅ is Q, L, N, [q], [l], [PE], [BhGln], [BhAsn], [aMeLeu],[aMeGln], [Bleu] or [BhLeu]; X₆ is R, [hArg], [r], [NMeArg], [NMehArg],[rhArg], [rArg] or [BhArg]; X₇ is P, [Sar], [Aib], [BhPro], [aMePro],[Oic], [rPro], or [Pip]; X₈ is R, [hArg], [r], [NMeArg], [NMehArg],[rhArg], [rArg] or [BhArg]; X₉ is L, [Cha], [NMeCha], [rCha], [rLeu],[NMeLeu], [aMeLeu], [BLeu], or [BhLeu]; X₁₀ is S, [aMeSer], [BhSer],[rSer], [Sar] or [bAla]; X₁₁ is H, A, V, L, Y, [Deg], [Tle], [NMeVal] or[bAla]; X₁₂ is K, [NMeLys], [BhLys], [BLys] or [bAla]; X₁₃ is G, [Sar],[Aib] or [bAla]; X₁₄ is P, [Sar], [Aib], [BhPro], [aMePro], [Oic],[Idc], [rPro], or [Pip]; X₁₅ is M, L, V, I, [Met(O)], [Nle], [Nva] or[pI-Phe]; X₁₆ is P, [Sar], [Aib], [BhPro], [aMePro], [Oic], [Idc],[rPro], or [Pip]; and X₁₇ is F, [Tic], [D-Tic], [Tiq], [D-Tiq],[4-Cl-F], [pI-Phe], [D-4FF], [D-4ClF], [D-4IF], [Idc], [Aic], [Oic],[D-Igl], [f], [D-1Nal], [D-2Nal], [1-Nal], [2-Nal] or [D-Bip] or isabsent. In one particular embodiment, X₁₆ is F, [Tic], [D-Tic], [Tiq],[D-Tiq], [4-Cl-F], [pI-Phe], [D-4FF], [D-4ClF], [D-4lF], [Idc], [Aic],[Oic], [D-Igl], [f], [D-1Nal], [D-2Nal], [1-Nal], [2-Nal] or [D-Bip] andX₁₇ is absent.

In certain embodiments, Z₁ is a C₁ to C₂₅ saturated or unsaturated fattyacyl group. For instance, Z₁ can be any fatty acyl group from Table 6,such as a fatty acyl group selected from Octanoyl (Oct) or Decanoyl(Dec) or Dodecanoyl (DDA), Tridecanoyl (TDA), Tetradecanoyl (Myristoyl),Pentadecanoyl (PDA), Hexadecanoyl (Palmitoyl), Heptadecanoyl (HDA),Octadecanoyl (Stearoyl), Octadecandioyl (ODDA). In some embodiments, Z₁is acetyl. In other embodiments, Z₁ is a 5 kDa, 10 kDa, or 20 kDa PEGpolymer or any other PEG polymer disclosed herein. In some embodiments,Z₂ is a conjugation linker comprising Aeea, γ-glutamate, or combinationsthereof. In other embodiments, the Z₂ conjugation linker is absent.

The present invention also provides for the use of one or more of themodified apelin polypeptides or conjugates thereof disclosed herein forthe treatment or prevention of a disease, disorder, or other medicalcondition, particularly cardiac disorders. In one embodiment, thepresent provides a method for treating a cardiovascular condition in asubject in need thereof comprising administering to the subject apharmaceutical composition comprising a therapeutically effective amountof any modified apelin polypeptide or half-life extension conjugatethereof described herein. A “therapeutically effective amount” refers tothe amount of a compound that, when administered to a subject fortreating a disease, or at least one of the clinical symptoms of adisease or disorder, is sufficient to affect such treatment for thedisease, disorder, or symptom. The “therapeutically effective amount”can vary depending on the specific polypeptide or peptide conjugateemployed, the disease, disorder, and/or symptoms of the disease ordisorder, severity of the disease, disorder, and/or symptoms of thedisease or disorder, the age of the subject to be treated, and/or theweight of the subject to be treated. An appropriate amount in any giveninstance can be readily apparent to those skilled in the art.

As used herein, “treating” or “treatment” of any disease or disorderrefers to arresting or ameliorating a disease, disorder, or at least oneof the clinical symptoms of a disease or disorder, reducing the risk ofacquiring a disease, disorder, or at least one of the clinical symptomsof a disease or disorder, reducing the development of a disease,disorder or at least one of the clinical symptoms of the disease ordisorder, or reducing the risk of developing a disease or disorder or atleast one of the clinical symptoms of a disease or disorder. “Treating”or “treatment” also refers to inhibiting the disease or disorder, eitherphysically, (e.g., stabilization of a discernible symptom),physiologically, (e.g., stabilization of a physical parameter), or both,or inhibiting at least one physical parameter which may not bediscernible to the subject. Further, “treating” or “treatment” refers todelaying the onset of the disease or disorder or at least symptomsthereof in a subject which may be exposed to or predisposed to a diseaseor disorder even though that subject does not yet experience or displaysymptoms of the disease or disorder.

The cardiovascular conditions that may be treated or ameliorated withthe modified apelin polypeptides or peptide conjugates of the inventioninclude, but are not limited to, disorders affecting contractility ofthe heart or any form of heart disease or disorders accompanied by heartdysfunction, such as cardiac hypertrophy, hypertension, heart failure,congestive heart failure, cardiogenic shock, septic shock, acutedecompensatory failure, pulmonary hypertension, myocardial infarction,ischemia, ischemia reperfusion injury, or cardiomyopathy. In certainembodiments, the cardiovascular condition is heart failure. All forms ofheart failure, including but not limited to, systolic failure, diastolicfailure, right ventricular failure, chronic heart failure, acute heartfailure, and decompensated congestive heart failure, may be treated orameliorated according to the methods of the invention. The modifiedapelin polypeptides of the invention or conjugates thereof can be usedto treat patients with reduced ejection fraction or preserved ejectionfraction. In one embodiment, the heart failure to be treated accordingto the methods of the invention is chronic systolic heart failure. Inanother embodiment, the heart failure to be treated according to themethods of the invention is chronic diastolic heart failure. In yetanother embodiment, the heart failure to be treated according to themethods of the invention is acute heart failure. In some embodiments,the cardiovascular condition to be treated according to the methods ofthe invention is hypertension.

In other embodiments, the present invention provides a method ofimproving cardiac contractility in a subject having a cardiovascularcondition comprising administering to the subject a pharmaceuticalcomposition comprising a therapeutically effective amount of anymodified apelin polypeptide or half-life extension conjugate thereofdescribed herein, wherein cardiac contractility is improved followingadministration. In certain embodiments, the subject has chronic or acuteheart failure. In some embodiments, administration of the pharmaceuticalcomposition increases dP/dt max or ejection fraction as compared tothese parameters prior to administration of the composition or to theseparameters in a subject not receiving the pharmaceutical composition.Administration of the pharmaceutical composition may also result inincreased exercise capacity, increased cardiac ejection fraction,decreased left ventricular end diastolic pressure, decreased pulmonarycapillary wedge pressure, increased cardiac output, increased cardiacindex, lowered pulmonary artery pressures, decreased left ventricularend systolic and diastolic dimensions, decreased left and rightventricular wall stress, and decreased wall tension relative to theseparameters prior to administration of the pharmaceutical composition orto these parameters in a subject not receiving the pharmaceuticalcomposition.

In one embodiment, the present invention also provides a method forincreasing ejection fraction in a subject having a cardiovascularcondition comprising administering to the subject a pharmaceuticalcomposition comprising a therapeutically effective amount of anymodified apelin polypeptide or half-life extension conjugate thereofdescribed herein, wherein ejection fraction is increased followingadministration. In certain embodiments, the subject has acute or chronicheart failure.

In some embodiments, the subject to be treated according to the methodsof the invention has one or more risk factors for heart diseaseincluding, for example, long standing uncontrolled hypertension,uncorrected valvular disease, chronic angina, recent myocardialinfarction, congestive heart failure, congenital predisposition to heartdisease or pathological hypertrophy. Alternatively or in addition, thesubject may have been diagnosed as having a genetic predisposition to,for example, cardiac hypertrophy, hypertension, or other cardiacdisorder or may have a familial history of, for example, cardiachypertrophy, hypertension, or other cardiac disorder.

Other embodiments of the invention concern the use of the modifiedapelin polypeptides or conjugates thereof described herein to treatcardiac symptoms resulting from pulmonary arterial hypertension, cancer,diabetes, obesity, metastatic diseases, and HIV. Other embodimentsconcern the use of the modified apelin polypeptides or conjugatesthereof described herein for the treatment or prevention of variousother disorders or diseases, such as, but not limited, to treatment ofdisorders associated with lipid and glucose metabolism, disordersassociated with regulation and function of the GI tract, protectionagainst HIV cell entry, and protection from cell apoptosis.

Pharmaceutical compositions comprising a modified apelin polypeptide orconjugate thereof can be configured for administration to a patient by awide variety of delivery routes, e.g., an intravascular delivery routesuch as by injection or infusion, subcutaneous (“s.c.”), intravenous(“i.v.”), intramuscular, intraperitoneal (“i.p.”), epidural, orintrathecal delivery routes, or for oral, enteral, pulmonary (e.g.,inhalant), intranasal, transmucosal (e.g., sublingual administration),transdermal or other delivery routes and/or forms of administrationknown in the art. Delivery of a drug or pharmaceutical compositioncontaining a modified apelin polypeptide or conjugate thereof, may takeplace via standard injectable modalities, whether self-administeredusing prefilled syringes or in hospital setting, or also via a deliverypump such as an autoinjector, a patch pump or large volume injector toachieve the most accurate dosing and the most stable plasma exposurelevels.

Thus, the present invention also includes pharmaceutical compositionscomprising any of the modified apelin polypeptides or conjugates thereofdescribed herein and a pharmaceutically acceptable carrier. Thepharmaceutical compositions may be prepared in liquid form, or may be indried powder form, such as lyophilized form. For oral or enteral use,the pharmaceutical compositions can be configured, for example, astablets, troches, lozenges, aqueous or oily suspensions, dispersiblepowders or granules, emulsions, hard or soft capsules, syrups, elixirsor enteral formulas.

In the practice, the “pharmaceutically acceptable carrier” can be anyphysiologically tolerated substance known to those of ordinary skill inthe art useful in formulating pharmaceutical compositions, including,any pharmaceutically acceptable diluents, excipients, dispersants,binders, fillers, glidants, anti-frictional agents, compression aids,tablet-disintegrating agents (disintegrants), suspending agents,lubricants, flavorants, odorants, sweeteners, permeation or penetrationenhancers, preservatives, surfactants, solubilizers, emulsifiers,thickeners, adjuvants, dyes, coatings, encapsulating material(s), and/orother additives singly or in combination. Such pharmaceuticalcompositions can include diluents of various buffer content (e.g.,Tris-HCl, acetate, phosphate), pH and ionic strength; additives such asdetergents and solubilizing agents (e.g., Tween® 80, Polysorbate 80),anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives(e.g., Thimersol®, benzyl alcohol) and bulking substances (e.g.,lactose, mannitol); incorporation of the material into particulatepreparations of polymeric compounds such as polylactic acid,polyglycolic acid, etc. or into liposomes. Hyaluronic acid can also beused, and this can have the effect of promoting sustained duration inthe circulation. Such compositions can influence the physical state,stability, rate of in vivo release, and rate of in vivo clearance of thepresent proteins and derivatives. See, e.g., Remington's PharmaceuticalSciences, 18th ed., Mack Publishing Co., Easton, Pa. 18042, pp.1435-1712, 1990, which are herein incorporated by reference in theirentirety. The compositions can be prepared in liquid form, or can be indried powder, such as lyophilized form. Implantable sustained releaseformulations are also useful, as are transdermal or transmucosalformulations. Additionally (or alternatively), various embodimentsprovide compositions for use in any of the various slow or sustainedrelease formulations or microparticle formulations known to the skilledartisan, for example, sustained release microparticle formulations,which can be administered via pulmonary, intranasal, or subcutaneousdelivery routes. (See, e.g., Murthy et al., “Injectable compositions forthe controlled delivery of pharmacologically active compound”, U.S. Pat.No. 6,887,487; Manning et al., “Solubilization of pharmaceuticalsubstances in an organic solvent and preparation of pharmaceuticalpowders using the same”, U.S. Pat. Nos. 5,770,559 and 5,981,474;Lieberman et al., “Lipophilic complexes of pharmacologically activeinorganic mineral acid esters of organic compounds”, U.S. Pat. No.5,002,936; Gen, “Formative agent of protein complex”, US 2002/0119946A1; Goldenberg et al., “Sustained release formulations”, WO 2005/105057A1).

One can dilute the compositions or increase the volume of thepharmaceutical compositions with an inert material. Such diluents caninclude carbohydrates, especially, mannitol, α-lactose, anhydrouslactose, cellulose, sucrose, modified dextrans and starch. Certaininorganic salts may also be used as fillers, including calciumtriphosphate, magnesium carbonate and sodium chloride.

A variety of conventional thickeners are useful in creams, ointments,suppository and gel configurations of the pharmaceutical composition,such as, but not limited to, alginate, xanthan gum, or petrolatum, mayalso be employed in various configurations of the pharmaceuticalcomposition.

In various embodiments, liquid pharmaceutical compositions that aresterile solutions or suspensions can be administered to a patient byinjection, for example, intramuscularly, intrathecally, epidurally,intravascularly (e.g., intravenously or intraarterially),intraperitoneally or subcutaneously. See, e.g., Goldenberg et al.,“Suspensions for the sustained release of proteins”, U.S. Pat. No.6,245,740 and WO 00/38652 A1. Sterile solutions can also be administeredby intravenous infusion. The composition can be included in a sterilesolid pharmaceutical composition, such as a lyophilized powder, whichcan be dissolved or suspended at a convenient time before administrationto a patient using sterile water, saline, buffered saline or otherappropriate sterile injectable medium.

Implantable sustained release formulations are also useful embodimentsof the pharmaceutical compositions. For example, the pharmaceuticallyacceptable carrier, being a biodegradable matrix implanted within thebody or under the skin of a human or non-human vertebrate, can be ahydrogel similar to those described above. Alternatively, it may beformed from a poly-alpha-amino acid component. (Sidman, “Biodegradable,implantable drug delivery device, and process for preparing and usingsame”, U.S. Pat. No. 4,351,337). Other techniques for making implantsfor delivery of drugs are also known and useful.

In powder forms, the pharmaceutically acceptable carrier is a finelydivided solid, which is in admixture with finely divided activeingredient(s), including the composition. For example, in someembodiments, a powder form is useful when the pharmaceutical compositionis configured as an inhalant. See, e.g., WO 2004/017918; U.S. Pat. No.6,900,317).

One can dilute or increase the volume of the compound with an inertmaterial. These diluents could include carbohydrates, especiallymannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans and starch. Certain inorganic salts can also be used as fillersincluding calcium triphosphate, magnesium carbonate and sodium chloride.Some commercially available diluents are Fast-Flo™, Emdex™, STA-Rx™1500, Emcompress™ and Avicell™

Binders can be used to hold the therapeutic agent together to form ahard tablet and include materials from natural products such as acacia,tragacanth, starch and gelatin. Others include methyl cellulose (MC),ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic. Anantifrictional agent can be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants can be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantscan also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, Carbowax 4000and 6000.

Also useful in various embodiments are oral dosage forms. If necessary,the composition can be chemically modified so that oral delivery isefficacious. Generally, the chemical modification contemplated is theattachment of at least one moiety to the molecule itself, where saidmoiety permits (a) inhibition of proteolysis; and (b) uptake into theblood stream from the stomach or intestine. Also desired is the increasein overall stability of the compound and increase in circulation time inthe body. Moieties useful as covalently attached half-life extendingmoieties can also be used for this purpose. Examples of such moietiesinclude: PEG, copolymers of ethylene glycol and propylene glycol,carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone and polyproline. See, for example, Abuchowski and Davis,“Soluble Polymer-Enzyme Adducts, Enzymes as Drugs”, Hocenberg andRoberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-383, 1981;Newmark et al., J. Appl. Biochem., 4:185-189, 1982. Other polymers thatcould be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferredfor pharmaceutical usage, as indicated above, are PEG moieties.

For oral delivery dosage forms, it is also possible to use a salt of amodified aliphatic amino acid, such as sodiumN-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), as a carrier to enhanceabsorption of the therapeutic compounds. The clinical efficacy of aheparin formulation using SNAC has been demonstrated in a Phase II trialconducted by Emisphere Technologies. See U.S. Pat. No. 5,792,451, “Oraldrug delivery composition and methods.”

In one embodiment, the pharmaceutically acceptable carrier can be aliquid and the pharmaceutical composition is prepared in the form of asolution, suspension, emulsion, syrup, elixir or pressurizedcomposition. The active ingredient(s) (e.g., the composition of matter)can be dissolved, diluted or suspended in a pharmaceutically acceptableliquid carrier such as water, an organic solvent, a mixture of both, orpharmaceutically acceptable oils or fats. The liquid carrier can containother suitable pharmaceutical additives such as detergents and/orsolubilizers (e.g., Tween 80, Polysorbate 80), emulsifiers, buffers atappropriate pH (e.g., Tris-HCl, acetate, phosphate), adjuvants,anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives(e.g., Thimersol, benzyl alcohol), sweeteners, flavoring agents,suspending agents, thickening agents, bulking substances (e.g., lactose,mannitol), colors, viscosity regulators, stabilizers, electrolytes,osmolutes or osmo-regulators. Additives can also be included in theformulation to enhance uptake of the composition. Additives potentiallyhaving this property are for instance the fatty acids oleic acid,linoleic acid and linolenic acid.

Also useful are oral solid dosage forms, which are described generallyin Remington's Pharmaceutical Sciences (1990), supra, in Chapter 89,which is hereby incorporated by reference in its entirety. Solid dosageforms include tablets, capsules, pills, troches or lozenges, cachets orpellets. Also, liposomal or proteinoid encapsulation can be used toformulate the present compositions (as, for example, proteinoidmicrospheres reported in U.S. Pat. No. 4,925,673). Liposomalencapsulation can be used and the liposomes can be derivatized withvarious polymers (e.g., U.S. Pat. No. 5,013,556). A description ofpossible solid dosage forms for the therapeutic is given in Marshall,K., Modern Pharmaceutics (1979), edited by G. S. Banker and C. T.Rhodes, in Chapter 10, which is hereby incorporated by reference in itsentirety. In general, the formulation will include the compound, andinert ingredients that allow for protection against the stomachenvironment, and release of the biologically active material in theintestine.

In tablet form, the active ingredient(s) are mixed with apharmaceutically acceptable carrier having the necessary compressionproperties in suitable proportions and compacted in the shape and sizedesired.

The powders and tablets can contain up to 99% of the activeingredient(s). Suitable solid carriers include, for example, calciumphosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch,gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ionexchange resins.

Controlled release formulation can be desirable. In various embodimentsthe composition can be incorporated into an inert matrix that permitsrelease by either diffusion or leaching mechanisms e.g., gums. Slowlydegenerating matrices can also be incorporated into the formulation,e.g., alginates, polysaccharides. Another form of a controlled releaseof the compositions is by a method based on the Oros™ therapeutic system(Alza Corp.), i.e., the drug is enclosed in a semipermeable membranewhich allows water to enter and push drug out through a single smallopening due to osmotic effects. Some enteric coatings also have adelayed release effect.

Pulmonary delivery of the compositions may also be useful. The protein(or derivative) is delivered to the lungs of a mammal while inhaling andtraverses across the lung epithelial lining to the blood stream. (Otherreports of this include Adjei et al., Pharma. Res. (1990) 7: 565-9;Adjei et al. (1990), Internal J. Pharmaceutics 63: 135-44 (leuprolideacetate); Braquet et al. (1989), J. Cardiovasc. Pharmacol. 13 (suppl.5): s. 143-146 (endothelin-1); Hubbard et al. (1989), Annals Int. Med.3: 206-12 (al-antitrypsin); Smith et al. (1989), J. Clin. Invest. 84:1145-6 (al-proteinase); Oswein et al. (March 1990), “Aerosolization ofProteins,” Proc. Symp. Resp. Drug Delivery II, Keystone, Colo.(recombinant human growth hormone); Debs et al. (1988), J. Immunol. 140:3482-8 (interferon-γ and tumor necrosis factor α) and Platz et al., U.S.Pat. No. 5,284,656 (granulocyte colony stimulating factor).

The dosage regimen involved in a method for treating a condition will bedetermined by the attending physician, considering various factors whichmodify the action of drugs, e.g. the age, condition, body weight, sexand diet of the patient, the severity of any infection, time ofadministration and other clinical factors. Generally, the daily regimenshould be in the range of 0.1-1000 micrograms of the compound perkilogram of body weight, preferably 0.1-150 micrograms per kilogram.

By way of further illustration, the following numbered embodiments arepresented:

Embodiment 1

An isolated polypeptide comprising the following formula:

(SEQ ID NO: 721) X₁ X₂ X₃ X₄

-   -   wherein:    -   X₁ is a fatty acyl group,    -   X₂ is γGlu or another suitable spacer moiety or is absent,    -   X₃ is a PEG group or another suitable spacer moiety or is        absent, and    -   X₄ is an apelin polypeptide.

In various embodiments, the modified apelin polypeptide of Embodiment 1does not have a half-life extension moiety (e.g PEG, lipid,immunoglobulin or immunoglobulin Fc), but will still have increasedstability relative to native apelin polypeptides and can have at least3, 4, 5, 6, 7 or 8 non-canonical amino acid substitutions.

Embodiment 2

The isolated polypeptide of embodiment 1 wherein X₂ is γGlu.

Embodiment 3

The isolated polypeptide of embodiment 1, wherein the fatty acyl groupis a C₁ to C₂₅ fatty acyl group.

Embodiment 4

The isolated polypeptide of embodiment 3, wherein the fatty acyl groupis Butanoyl, Hexanoyl, Octanoyl, Decanoyl, Dodecanoyl, Tridecanoyl,Tetradecanoyl, Pentadecanoyl, Hexadecanoyl, Heptadecanoyl, Octadecanoyl,Octadecandioyl, Octanedioyl, Decanedioyl, Dodecanedioyl, Hexanedioyl,Butanedioyl, Tetradecanedioyl, or Hexadecanedioyl.

Embodiment 5

The isolated polypeptide of any one of embodiments 1 to 4, wherein X₃ isAeea, Aeea-Aeea, γGlu-Aeea, γGlu-Aeea-Aeea or γGlu.

Embodiment 6

The isolated polypeptide of embodiment 1, wherein X₁ is Octadecandioyl,Heptadecanoyl, Tridecanoyl, Butanoyl, Hexanoyl, Hexadecanoyl,Butanedioyl, Octanedioyl, or Decanedioyl; X₂ is γ-Glu or is absent; andX₃ is Aeea, Aeea-Aeea or is absent.

Embodiment 7

The isolated polypeptide of any one of embodiments 1 to 6, wherein theapelin polypeptide is at least 12 amino acids long.

Embodiment 8

The isolated polypeptide of any one of embodiments 1 to 7, wherein theapelin polypeptide has at least one non-canonical amino acidsubstitution.

Embodiment 9

The isolated polypeptide of embodiment 8, wherein the apelin polypeptidehas 2, 3, 4, 5, 6, 7, 8 or 9 non-canonical amino acids.

Embodiment 10

The isolated polypeptide of embodiment 1, wherein the apelin polypeptidecomprises at least one D-amino acid, a β-amino acid, a non-canonicalamino acid, a N-methyl amino acid or an α-methyl amino acid, or the D-or β-form of the non-canonical amino acid.

Embodiment 11

The isolated polypeptide of embodiment 10, wherein the D-amino acid, theβ-amino acid, the N-methyl amino acid, the α-methyl amino acid, thenon-canonical amino acid or the D- or β-form of the non-canonical aminoacid replaces a canonical amino acid in full-length apelin (SEQ ID NO:2), apelin 42-77 (SEQ ID NO: 3), apelin 65-77 (apelin-13) (SEQ ID NO:4), apelin 61-77 (SEQ ID NO: 5) or fragments of full-length apelin.

Embodiment 12

An isolated polypeptide comprising the amino acid sequence:

(SEQ ID NO: 719) [z]X₁ X₂ X₃X₄ X₅ X₆ X₇SHX₈G[Oic] X₉ X₁₀ X₁₁,

z is acetyl or absent,

X₁ is acetyl, [r] or [hArg],

X₂ is [r], R or [hArg],

X₃ is Q or [q],

X₄ is [hArg], R or [r]

X₅ is P or [Oic],

X₆ is[r], [hArg] or [NMeArg],

X₇ is [NMeLeu] or[Cha],

X₈ is K or [NLysG],

X₉ is [pl-Phe] or [Nle]

X₁₀ is P or [D-1Nal] or [Pip], and

X₁₁ is a D-amino acid, a β-amino acid, a non-canonical amino acid or theD- or β-form of the non-canonical amino acid.

Embodiment 13

The isolated polypeptide of embodiment 12, wherein X₁₁ is [D-Bip],[4-CL-F], [D-4CLF], [TIC], [f] or is absent.

Embodiment 14

The isolated polypeptide of embodiment 12 having an amino acid sequenceselected from the group of sequences consisting of SEQ ID NOs: 16-44.

Embodiment 15

An isolated polypeptide comprising the amino acid sequence:{HDA}[AC4Abu][Aeea][Aeea]X₁X₂X₃P[Cha]SHKG[Oic][Nle]X₄X₅  (SEQ IDNO:722),wherein:

X₁ is [hArg] or is absent,

X₂ is Q or [q],

X₃ is [hArg] or [r],

X₄ is P or [Pip] and

X₅ is [4-Cl-F] or [D-4ClF]

Embodiment 16

The isolated polypeptide of embodiment 15 having an amino acid sequenceselected from the group of sequences consisting of SEQ ID NOs: 237, 239,or 242.

Embodiment 17

An isolated polypeptide comprising the amino acid sequence:

(SEQ ID NO: 720) [z] X₁ X₂[hArg][hArg]X₃X₄X₅X₆X₇ X₈X₉ X₁₀G[Oic][Nle]X₁₁[4-Cl—F],wherein:

z is acetyl,

X₁ is o or O,

X₂ is f or F,

X₃ is Q or [BLeu],

X₄ is [hArg] or [rhArg],

X₅ is P or [aMePro],

X₆ is [hArg] or [rhArg],

X₇ is [aMeLeu], [rCha], [BLeu] or [Cha],

X₈ is [NhSerG], [aMeS], [rSer], [DrSer], or S,

X₉ is H or [rHis]

X₁₀ is K, [NLysG], [rLys], or [aMeOrn], and

X₁₁ is P or [aMePro].

Embodiment 18

The isolated polypeptide of embodiment 17 having an amino acid sequenceselected from the group of sequences consisting of SEQ ID NOs: 29-44.

Embodiment 19

An isolated polypeptide comprising the amino acid sequence:{TDA}[AC4Abu][Aeea][Aeea]X₁X₂X₃X₄X₅X₆X₇X₈KG[Oic]X₉X₁₀X₁₁  (SEQ ID No.724),wherein:X₁ is [hArg], [r] or is absent,X₂ is Q, [q], [BLeu] or [NMeGln],X₃ is [hArg] or [r],X₄ is P, [Pip], [Oic] or [Sar],X₅ is [NMeArg], [r] or [hArg],X₆ is [Cha], [NMeLeu], [BLeu] or [NMeCha],X₇ is S, [BhSer], [bAla], [NhSerG] or [aMeS],X₈ is H, A, Y, [Tle], [Deg], L or V,X₉ is [Nle] or [pl-Phe],X₁₀ is P, a D-amino acid, a β-amino acid, a non-canonical amino acid orthe D- or β-form of the non-canonical amino acid, and X₁₁ is [4-Cl-F],[D-4ClF], [D-Bip] or is absent.

Embodiment 20

The isolated polypeptide of embodiment 19, wherein:

X₁₀ is [D-Tic], [4-Cl-F], [D-4ClF], [Aic], [Oic], [D-4F], [D-Ogl], [f],[1-Nal], [2-Nal], [D-Bip], [Tic], [Aib] or [Deg].

Embodiment 21

The isolated polypeptide of embodiment 19 having an amino acid sequenceselected from the group of sequences consisting of SEQ ID NOs: 121-210and 245-256.

Embodiment 22

The isolated polypeptide of embodiment 19, wherein TDA is replaced withHDA.

Embodiment 23

An isolated polypeptide comprising the amino acid sequence:{TDA}[AC4Abu][Aeea][Aeea]X₁q[hArg]P[NMeArg][Cha]SHKG[Oic][Nle]X₂X₃  (SEQID No. 725)wherein:

X₁ is [hArg] or is absent,

X₂ is P, a D-amino acid or a non-canonical amino acid, and

X₃ is a D-amino acid, a non-canonical amino acid or —COOH.

Embodiment 24

The isolated polypeptide of embodiment 23 having an amino acid sequenceselected from the group of sequences consisting of SEQ ID NOs: 121-152.

Embodiment 25

An isolated polypeptide comprising the amino acid sequence{TDA}[AC4Abu][Aeea][Aeea][hArg]q[hArg]P[NMeArg][Cha]SHKG[Oic][Nle]X₁COOH  (SEQID NO: 726),wherein X₁ is a D-amino acid, a β-amino acid, a non-canonical amino acidor the D- or β-form of the non-canonical amino acid.

Embodiment 26

The isolated polypeptide of embodiment 25, wherein X₁ is [D-Tic],[4-CL-F], [D-4ClF], [Aic], [Oic], [D-4lF], [D-IgL], [f], [1-Nal],[2-Nal], [D-Bip] or [Tic].

Embodiment 27

The isolated polypeptide of embodiment 25 having an amino acid sequenceselected from the group of sequences consisting of SEQ ID NOs: 166, 171,and 245-256.

Embodiment 28

An isolated polypeptide comprising the amino acid sequence{TDA}[AC4Abu][Aeea][Aeea]QX₁PX₂X₃SHKG[Oic]X₄X₅X₆  (SEQ ID No. 727),wherein:X₁ is R, r or [hArg],X₂ is r, [hArg] or [NMeArg],X₃ is [NMeLeu] or [Cha],X₄ is [pl-Phe] or [Nle],X₅ is P or [D-1Nal], andX₆ is [D-Bip], [4-CL-F], [D-4ClF] or is absent.

Embodiment 29

The isolated polypeptide of embodiment 28 having an amino acid sequenceselected from the group of sequences consisting of SEQ ID NOs: 165 and211-233.

Embodiment 30

An isolated polypeptide comprising the amino acid sequence

(SEQ ID No. 723) {HDA}[AC4Abu][Aeea][Aeea]X₁X₂X₃PX₄X₅SHKG[Oic][Nle] X₆X₇wherein:

X₁ is [hArg] or is absent,

X₂ is Q or [q],

X₃ is r or [hArg],

X₄ is r or [NMeArg],

X₅ is [NMeLeu] or [Cha],

X₆ is [Pip] or P, and

X₇ is [4-CL-F], [f], [D-4ClF], or [Tic].

Embodiment 31

The isolated polypeptide of embodiment 30, wherein HDA is replaced byTDA or other fatty acyl group.

Embodiment 32

The isolated polypeptide of embodiment 30 having an amino acid sequenceselected from the group of sequences consisting of SEQ ID NOs: 234-243.

Embodiment 33

An isolated polypeptide comprising the amino acid sequence

(SEQ ID No. 728) {TDA}[AC4Abu][Aeea][Aeea]X₁X₂X₃PX₄X₅X₆X₇KG[Oic] X₈X₉X₁₀wherein:

X₁ is [hArg] or [r],

X₂ is Q or [q],

X₃ is r or [hArg],

X₄ is r or [NMeArg],

X₅ is [NMeLeu][BLeu] or [Cha],

X₆ is S or [bAla],

X₇ is H, A or [Tle],

X₈ is [Nle] or [pl-Phe],

X₉ is P, a D-amino acid, a β-amino acid, a non-canonical amino acid orthe D- or β-form of the non-canonical amino acid, and

X₁₀ is [Oic], [D-4ClF], [D-1Nal], [D-Bip] or is absent.

Embodiment 34

The isolated polypeptide of embodiment 33, wherein X₉ is [D-Tic],[4-Cl-F], [D-4ClF], [Aic], [Oic], [D-igl], [f], [D-1Nal], [D-2Nal],[1-Nal], [2-Nal] or [D-Bip] and X₁₀ is absent.

Embodiment 35

The isolated polypeptide of embodiment 33, wherein TDA is replaced byHDA.

Embodiment 36

The isolated polypeptide of embodiment 33 having an amino acid sequenceselected from the group of sequences consisting of SEQ ID NOs: 153-155,166, 171, 173-175, 180, 199, 201, 208, and 245-256.

Embodiment 37

An isolated polypeptide comprising the amino acid sequence:

(SEQ ID No. 45) AcOF[hArg][hArg]Q[hArg]P[hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F].

Embodiment 38

An isolated polypeptide having an amino acid sequence selected from thegroup of any one of SEQ ID NOs: 7-714.

Embodiment 39

The isolated polypeptide of any one of embodiments 1 to 38, wherein thepolypeptide has increased stability relative to wild-type apelin 13 (SEQID NO: 4).

Embodiment 40

An isolated polypeptide comprising the formula:N₁[AC4Abu]N₂Xwherein:

N₁ is a fatty acyl group or Acetyl-NH,

N₂ is [Aeea][Aeea] or [Aeea], and

X is an apelin polypeptide of 11, 12, 13, 14, 15, 16 or 17 amino acidslong comprising E1xxK1 (cyclized via an amide bond between E and K)wherein xx can be 2-3 amino acids selected from D- or L-amino acids, α-or β-amino acids, homologated amino acids, N-methylated amino acids,α-methyl amino acids, amino acids bearing the side chain on N instead ofthe α-carbon or another canonical or non-canonical amino acid.

Embodiment 41

An isolated polypeptide comprising the amino acid sequence:ACX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇  (SEQ ID NO:718),wherein:

X₁ is O or [BhLys],

X₂ is F, [BhPhe] or [Bphe],

X₃ is [hArg], [NMeArg] or [BhArg],

X₄ is [hArg], [NMeArg] or [BhArg],

X₅ is Q, [BhGln], [BhAsn] or [BhLeu],

X₆ is [hArg], [NMeArg] or [BhArg],

X₇ is P, [Sar], [Aib], [BhPro] or [Pip],

X₈ is [hArg], [NMeArg] or [BhArg],

X₉ is [Cha] or [BhLeu],

X₁₀ is S, [BhSer], [Sar] or [bAla],

X₁₁ is H, [NMeVal] or [bAla],

X₁₂ is K, [NMeLys], [BhLys], [BLys] or [bAla],

X₁₃ is G, [Sar], [Aib] or [bAla],

X₁₄ is [Oic], [Aib], [Sar], [bAla], [BhPro] or [Pip],

X₁₅ is [Nle] or [bAla],

X₁₆ is P, [Sar], [Aib], [BhPro], [bAla], [Pip], [D-1Nal] or [D-2Nal] and

X₁₇ is [4-Cl-F], [Bh-Phe] or [BPhe].

Embodiment 42

The isolated polypeptide of embodiment 41 having an amino acid sequenceselected from the group of sequences consisting of SEQ ID NOs: 668-714.

Embodiment 43

An isolated polypeptide comprising the amino acid sequence:

(SEQ ID NO: 768) [Z]X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂ X₁₃PX₁₄

having only one amide bond between the carboxylate side chain of E andthe amino side chain of K and wherein:

Z is acetyl, acyl, [Atz(PEG10)], a PEG derivative,{TDA}[AC4Abu][Aeea][Aeea], or other half-life extension moiety or isabsent,

X₁ is r, [hArg], [NMeArg], R, absent, or E and if X₁ is E itscarboxylate sidechain forms an amide bond with the sidechain of a K atposition X₄ or X₈,

X₂ is r, [hArg], [NMeArg], R, absent, or E and if X₂ is E itscarboxylate sidechain forms an amide bond with the sidechain of a K atposition X₅ or X₆,

X₃ is Q, q, or E and if X₃ is E its carboxylate sidechain forms an amidebond with the sidechain of a K at position X₆ or X₇,

X₄ is r, [hArg], [NMeArg], R, K or E and if X₄ is E its carboxylatesidechain forms an amide bond with the sidechain of a K at position X₇or X₈, or if X₄ is K its amine sidechain forms an amide bond with thesidechain of E at position X₁,

X₅ is P, K or E and if X₅ is E its carboxylate sidechain forms an amidebond with the sidechain of a K at position X₈ or X₉, or if X₅ is K itsamine sidechain forms an amide bond with the sidechain of E at positionX₁ or X₂,

X₆ is r, [hArg], [NMeArg], R, K or E and if X₆ is E its carboxylatesidechain forms an amide bond with the sidechain of a K at position X₉or X₁₀, or if X₆ is K its amine sidechain forms an amide bond with thesidechain of E at position X₂ or X₃,

X₇ is [Cha], [NMeLeu], K or E and if X₇ is E its carboxylate sidechainforms an amide bond with the sidechain of a K at position X₁₀ or X₁₁, orif K is X₇ its amine sidechain forms an amide bond with the sidechain ofE at position X₃ or X₄,

X₈ is S, K or E and if X₈ is E its carboxylate sidechain forms an amidebond with the sidechain of a K at position X₁₀ or X₁₁, or if X₈ is K itsamine sidechain forms an amide bond with the sidechain of E at positionX₄ or X₈,

X₉ is H, or K and if X₉ is E its amine sidechain forms an amide bondwith the sidechain of E at position X₅ or X₆,

X₁₀ is K and its amine sidechain can, but does not necessarily form anamide bond with the sidechain of E at position X₆ or X₇,

X₁₁ is G, or K and if X₁₁ is K its amine sidechain forms an amide bondwith the sidechain of E at position X₇ or X₈,

X₁₂ is [Oic], or K and if X₁₂ is K its amine sidechain forms an amidebond with the sidechain of E at position X₈ or X₉,

X₁₃ is [p-I-Phe], [Nle], or K and if X₁₃ is E its amine sidechain formsan amide bond with the sidechain of E at position X₉, and

X₁₅ is [D-Bip], [D-4ClF] or [4-Cl-F].

Embodiment 44

An isolated polypeptide comprising the amino acid sequence:Z₁Z₂X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇  (SEQ ID NO:769),wherein

Z₁ is an acyl group

Z₂ comprises a conjugation linker or is absent,

X₁ is O, K, [D-Orn], [k], [BLys], [D-BLys], [BhLys], or [D-BhLys] or isabsent,

X₂ is F, [BhPhe], [BPhe] or is absent,

X₃ is R, [hArg], [r], [NMeArg], [NMehArg], [rhArg], [rArg], [BhArg] oris absent,

X₄ is R, [hArg], [r], [NMeArg], [NMehArg], [rhArg], [rArg], [BhArg] oris absent,

X₅ is Q, L, N, [q], [l], [PE], [BhGln], [BhAsn], [aMeLeu], [aMeGln],[Bleu] or [BhLeu],

X₆ is R, [hArg], [r], [NMeArg], [NMehArg], [rhArg], [rArg] or [BhArg],

X₇ is P, [Sar], [Aib], [BhPro], [aMePro], [Oic], [rPro], or [Pip],

X₈ is R, [hArg], [r], [NMeArg], [NMehArg], [rhArg], [rArg] or [BhArg],

X₉ is L, [Cha], [NMeCha], [rCha], [rLeu], [NMeLeu], [aMeLeu], [BLeu], or[BhLeu],

X₁₀ is S, [aMeSer], [BhSer], [rSer], [Sar] or [bAla],

X₁₁ is H, A, V, L, Y, [Deg], [Tle], [NMeVal] or [bAla],

X₁₂ is K, [NMeLys], [BhLys], [BLys] or [bAla],

X₁₃ is G, [Sar], [Aib] or [bAla],

X₁₄ is P, [Sar], [Aib], [BhPro], [aMePro], [Oic], [Idc], [rPro], or[Pip],

X₁₅ is M, L, V, I, [Met(O)], [Nle], [Nva] or [pI-Phe],

X₁₆ is P, [Sar], [Aib], [BhPro], [aMePro], [Oic], [Idc], [rPro], or[Pip], and

X₁₇ is F, [Tic], [D-Tic], [Tiq], [D-Tiq], [4-Cl-F], [pI-Phe], [D-4FF],[D-4ClF], [D-4lF], [Idc], [Aic], [Oic], [D-Igl], [f], [D-1Nal],[D-2Nal], [1-Nal], [2-Nal] or [D-Bip] or is absent.

Embodiment 45

The isolated polypeptide of embodiment 44, wherein Z₁ is a C₁ to C₂₅saturated or unsaturated fatty acyl group.

Embodiment 46

The isolated polypeptide of embodiment 44, wherein Z₁ is Acetyl,Octanoyl (Oct), Decanoyl (Dec), Dodecanoyl (DDA), Tridecanoyl (TDA),Tetradecanoyl (Myristoyl), Pentadecanoyl (PDA), Hexadecanoyl(Palmitoyl), Heptadecanoyl (HDA), Octadecanoyl (Stearoyl),Octadecandioyl (ODDA) or any fatty acyl or lipophilic group incorporatedto extend the half-life of the polypeptide.

Embodiment 47

The isolated polypeptide of embodiment 44, wherein Z₁ is {5 kDa}, {10kDa}, {20 kDa} or any other polyethylene glycol (PEG) polymer that isincorporated to extend the half-life of the polypeptide.

Embodiment 48

The isolated polypeptide of embodiment 44, wherein Aeea, γ-glutamate, orany other moiety is present as a constituent of the Z₂ conjugationlinker.

Embodiment 49

The isolated polypeptide of embodiment 48, wherein the conjugationlinker may be a polar, non-polar, hydrophobic, aliphatic, or aromaticmoiety that serves a special, functional or structural role.

Embodiment 50

The isolated polypeptide of embodiment 44, wherein the Z₂ conjugationlinker is absent.

Embodiment 51

The isolated polypeptide of embodiment 44, wherein X₁₆ is F, [Tic],[D-Tic], [Tiq], [D-Tiq], [4-Cl-F], [pI-Phe], [D-4FF], [D-4ClF], [D-4lF],[Idc], [Aid], [Oic], [D-Igl], [f], [D-1 Nal], [D-2Nal], [1-Nal], [2-Nal]or [D-Bip] and X₁₇ is absent.

Embodiment 52

An isolated polypeptide comprising the amino acid sequence:X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀GX₁₁X₁₂X₁₃X₁₄  (SEQ ID NO:717),wherein:

X₁ is R, E, [hArg], or absent;

X₂ is [r], R, E, [hArg], or absent;

X₃ is Q, [q], or [BLeu];

X₄ is [hArg], [NMeArg], R, E, or [r];

X₅ is P or [aMePro];

X₆ is R, E, [r], [hArg] or [NMeArg];

X₇ is L, [aMeLeu], [BLeu], [NMeLeu] or [Cha];

X₈ is S, [BhSer], or [NhSerG];

X₉ is H or Y;

X₁₀ is K or [NLysG];

X₁₁ is P, [Oic], [aMePro], or [Pip];

X₁₂ is [Nle], [rNle], or [pI-Phe];

X₁₃ is P, [BhPro], [aMePro], or [Aib]; and

X₁₄ is F, [D-BhPhe], [4-Cl-F], [D-4ClF], or [D-Bip].

Embodiment 53

The isolated polypeptide of embodiment 52, wherein the polypeptide isacetylated at its amino terminus.

Embodiment 54

The isolated polypeptide of embodiment 52, wherein the polypeptide isconjugated to a C₁ to C₂₅ saturated or unsaturated fatty acyl groupoptionally through a conjugation linker.

Embodiment 55

The isolated polypeptide of embodiment 54, wherein the fatty acyl groupis Tridecanoyl, Butanoyl, Hexanoyl, Hexadecanoyl, Butanedioyl,Octanedioyl, or Decanedioyl.

Embodiment 56

The isolated polypeptide of embodiment 54, wherein the fatty acyl groupis Octanoyl, Decanoyl, Dodecanoyl, Tridecanoyl, Tetradecanoyl,Pentadecanoyl, Hexadecanoyl, Heptadecanoyl, Octadecanoyl, orOctadecandioyl.

Embodiment 57

The isolated polypeptide of embodiment 54, wherein the conjugationlinker comprises Aeea, Aeea-Aeea, γGlu-Aeea, γGlu-Aeea-Aeea, or γGlu.

Embodiment 58

The isolated polypeptide of embodiment 52, wherein the polypeptide isconjugated to a polyethylene glycol (PEG) polymer optionally through aconjugation linker.

Embodiment 59

The isolated polypeptide of embodiment 58, wherein the PEG polymer is a5 kDa, 10 kDa, or 20 kDa PEG polymer.

Embodiment 60

The isolated polypeptide of embodiment 58, wherein the conjugationlinker comprises 3-mercaptopropanoic acid.

Embodiment 61

The isolated polypeptide of embodiment 52, wherein the polypeptide isconjugated to an immunoglobulin or an immunoglobulin Fc domainoptionally through a conjugation linker.

Embodiment 62

The isolated polypeptide of embodiment 61, wherein the conjugationlinker is a peptidyl linker.

Embodiment 63

The isolated polypeptide of embodiment 61, wherein the conjugationlinker is a non-peptidyl linker.

Embodiment 64

The isolated polypeptide of embodiment 63, wherein the non-peptidyllinker comprises a PEG polymer.

Embodiment 65

The isolated polypeptide of embodiment 52, wherein X₇ is [NMeLeu], X₁₂is [pI-Phe], and X₁₄ is [D-Bip].

Embodiment 66

The isolated polypeptide of embodiment 52, wherein X₁ is [hArg], X₂ is[hArg], X₃ is Q, X₄ is [hArg], and X₅ is P.

Embodiment 67

The isolated polypeptide of embodiment 52, wherein X₆ and X₇ are[NMeArg] and [aMeLeu], [hArg] and [BLeu], or [hArg] and [aMeLeu].

Embodiment 68

The isolated polypeptide of embodiment 52, wherein X₁₃ is [BhPro],[aMePro], or [Aib] and X₁₄ is [D-BhPhe] or [4-Cl-F].

Embodiment 69

The isolated polypeptide of embodiment 52, wherein the polypeptidecomprises the amino acid sequence selected from SEQ ID NOs: 8-11, 16,17, 31, 32, 45, 53, 60, 68, 69-71, 92, 112, 114, 119, 120, 221, 228,237, 263, 286, 287, 362, 373, 376, 379, 382, 388, 412, 416, 460, 468,482, 483, 485, 491, 498, 499, 500, 502, 505, 514, 519, 526, 531, 534,544, 552, 554, 560, and 571.

Embodiment 70

A pharmaceutical composition comprising the polypeptide of any one ofembodiments 1 to 69 and a pharmaceutically acceptable carrier.

Embodiment 71

A method for treating a cardiovascular condition in a subject in needthereof comprising administering to the subject an isolated polypeptideof any one of embodiments 1 to 69.

Embodiment 72

The method of embodiment 71, wherein the cardiovascular condition isheart failure.

Embodiment 73

The method of embodiment 72, wherein the heart failure is heart failurewith reduced ejection fraction.

Embodiment 74

The method of embodiment 72, wherein the heart failure is heart failurewith preserved ejection fraction.

Embodiment 75

The method of embodiment 72, wherein the heart failure is chronicsystolic heart failure or chronic diastolic heart failure.

Embodiment 76

The method of embodiment 72, wherein the heart failure is acute heartfailure. Embodiment 77.

The method of embodiment 71, wherein the cardiovascular condition ishypertension.

Embodiment 78

A method of improving cardiac contractility in a subject having acardiovascular condition comprising administering to the subject thepolypeptide of any one of embodiments 1 to 69, wherein cardiaccontractility is improved in the subject following administration.

Embodiment 79

The method of embodiment 78, wherein the cardiovascular condition isheart failure.

Embodiment 80

The method of embodiment 79, wherein the heart failure is chronicsystolic heart failure or chronic diastolic heart failure.

Embodiment 81

The method of any one of embodiments 71 to 80, wherein dP/dt max and/orejection fraction is increased in the subject following administrationof the polypeptide.

Embodiment 82

The method of any one of embodiments 71 to 80, wherein the systolic ordiastolic function is improved in the subject following administrationof the polypeptide.

Embodiment 83

A method of increasing ejection fraction in a subject having acardiovascular condition comprising administering to the subject thepolypeptide of any one of embodiments 1 to 69, wherein the ejectionfraction is increased following administration of the polypeptide.Embodiment 84.

The method of embodiment 83, wherein the cardiovascular condition isheart failure.

Embodiment 85

The method of embodiment 84, wherein the heart failure is chronicsystolic heart failure or chronic diastolic heart failure.

Embodiment 86

A method of improving systolic or diastolic function in a subject havinga cardiovascular condition comprising administering to the subject thepolypeptide of any one of embodiments 1 to 69.

Embodiment 87

The method of embodiment 86, wherein the cardiovascular condition isheart failure.

Embodiment 88

The method of embodiment 87, wherein the heart failure is chronicsystolic heart failure or chronic diastolic heart failure.

Embodiment 89

A method of treating cardiac failure in a patient in need thereofcomprising administering to the patient the polypeptide of any one ofembodiments 1 to 69.

Embodiment 90

The method of embodiment 89, wherein the cardiac failure is cardiacfailure with reduced ejection fraction.

Embodiment 91

The method of embodiment 89, wherein the cardiac failure is cardiacfailure with preserved ejection fraction.

Embodiment 92

The method of embodiment 89, wherein the cardiac failure is chronicsystolic cardiac failure or chronic diastolic cardiac failure.

The previous description and the following working examples areillustrative and not to be construed in any way as limiting the scope ofthe invention.

EXAMPLES Example 1. Preparation of Modified Apelin Polypeptides

Polypeptides have been prepared that can act as APJ agonists. Thepolypeptides were screened for potency and metabolic stability asdescribed in detail in the following examples. The sequences wereoptimized through techniques known in the art in which one or more aminoacids may be changed while maintaining or improving the affinity,functional activity, or stability of the peptide. Nishizawa et al.,Peptide Science 37:151-157, 2001; Murza et al., Chem MeD Chem.,7:318-325, 2012.

Various non-canonical amino acids were used in the preparation of themodified apelin polypeptides. Some of these amino acids are availablecommercially. The following describes methods for making certainnon-canonical amino acids that can be used in the synthesis of modifiedapelin polypeptides.

Preparation of Compound 1

4-Methylmorpholine (1.25 mL, 11.4 mmol) and ethyl chloroformate (1.09mL, 11.4 mmol) were added to a stirred solution of(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(1,3-bis(tert-butoxycarbonyl)guanidino)hexanoicacid (6.95 g, 11.4 mmol) in THF (50 mL) at −10° C. in a 3-neck flaskequipped with a thermometer. The mixture was stirred at −10° C. for 10min. Sodium borohydride (1.29 g, 34.1 mmol) was added, and the reactionmixture was warmed to 0° C. Methanol (50 mL) was then added slowly at 0°C. over 30 min via addition funnel. The reaction mixture was thendiluted with ethyl acetate and washed with 5% aqueous citric acid. Theaqueous layer was separated and extracted once more with ethyl acetate.The combined organic layers were washed with 5% aqueous citric acid,saturated aqueous sodium bicarbonate, saturated aqueous sodium chloride,dried over anhydrous magnesium sulfate, filtered, and concentrated invacuo to give an oil. The oil was purified by silica gel chromatography(50 to 100% EtOAc/heptane gradient) to give 1 (4.68 g, 69% yield) as awhite solid. LC/MS (ESI⁻) m/z=597.3 (M+H)⁺.

Preparation of Compound 2

Dess-martin periodinane (5.19 g, 12.2 mmol) was added to an ice coldsolution of 1 (5.62 g, 9.42 mmol) in DCM (50 mL) under argon. The icebath was removed and the mixture was stirred for 3 h. The mixture waspoured into a mixture of diethyl ether (100 mL) and saturated aqueoussodium bicarbonate (100 mL), and then it was stirred vigorously for 30min. The resulting suspension was filtered and the filter cake waswashed with diethyl ether. The biphasic filtrate was separated and theaqueous layer was extracted with diethyl ether (2×). The combinedextracts were dried over anhydrous magnesium sulfate, filtered, andconcentrated in vacuo to give 2, which was used without furtherpurification.

Preparation of Compound 3

1-(−)-Proline (2.71 g, 23.6 mmol) and sodium triacetoxyborohydride (2.99g, 14.1 mmol) were added to a 0° C. solution of 2 in DCM (50 mL) and themixture was stirred overnight with warming to RT. Saturated aqueoussodium bicarbonate was added and the mixture was stirred until thebiphasic mixture became clear. The layers were separated and the aqueouslayer was extracted with 9:1 DCM/MeOH (2×). The combined extracts weredried over anhydrous magnesium sulfate, filtered, and concentrated invacuo to give on oil. The oil was purified by silica gel chromatography(DCM to 9% MeOH/1% AcOH in DCM gradient) to give(S)-1-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(1,3-bis(tert-butoxycarbonyl)guanidino)hexyl)pyrrolidine-2-carboxylicacid (3, 2.14 g, 33% yield from 1) as a white solid. LC/MS (ESI⁻)m/z=694.4 (M+H)⁺.

Preparation of Compound 5

N,N′-Dicyclohexylcarbodiimide (2.04 g, 9.90 mmol) and fmoc-lys(boc)-OH(4.22 g, 9.00 mmol) were added to a stirred mixture ofN,O-dimethylhydroxylamine hydrochloride (0.97 g, 9.90 mmol) andtriethylamine (1.38 mL, 9.90 mmol) in DCM (40 mL). The reaction mixturewas stirred at RT for 2.5 h. The reaction mixture was cooled to −20° C.and filtered. The collected solid was washed with cold DCM and thendiscarded. The filtrate was washed with saturated aqueous sodiumbicarbonate, dried over anhydrous magnesium sulfate, filtered, andconcentrated in vacuo. The resulting crude product was purified viasilica gel chromatography, eluting with 10% to 75% EtOAc in heptane, togive (S)-(9H-fluoren-9-yl)methyl tert-butyl(6-(methoxy(methyl)amino)-6-oxohexane-1,5-diyl)dicarbamate (5, 4.31 g,91% yield) as an oil. LC/MS (ESI⁻) m/z=534.2 (M+Na)⁺.

Preparation of Compound 6

Lithium aluminum hydride (1.0M solution in THF, 3.29 mL, 3.29 mmol) wasadded dropwise to a solution of (S)-(9H-fluoren-9-yl)methyl tert-butyl(6-(methoxy(methyl)amino)-6-oxohexane-1,5-diyl)dicarbamate (5, 886 mg,1.73 mmol) in THF (9 mL) at 0° C. This mixture was stirred for 45 min.before being quenched at 0° C. by the careful addition of aqueous 1Mpotassium bisulfate solution. The resulting biphasic mixture wasseparated and the aqueous layer was extracted with ethyl acetate (1×).The combined extracts were washed with water (1×), dried over anhydrousmagnesium sulfate, filtered, and concentrated in vacuo to give (S)-allyl2-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexyl)amino)acetate(6) as an oil. This material was taken to the next step without furtherpurification.

Preparation of Compound 7

(S)-allyl2-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexyl)amino)acetate(compound 6) was dissolved in DCM (4 mL) and cooled to 0° C. A solutionof allyl 2-aminoacetate (0.20 g, 1.73 mmol) in DCM (1 mL) was addeddropwise followed by sodium triacetoxyborohydride (0.44 g, 2.08 mmol).This mixture was stirred at 0° C. for 1 h, then quenched with saturatedaqueous sodium bicarbonate. The resulting biphasic mixture was separatedand the aqueous layer was extracted with DCM (1×). The combined organicextracts were dried over anhydrous magnesium sulfate, filtered, andconcentrated in vacuo to give an oil that was purified by silica gelchromatography (50 to 100% EtOAc/heptane gradient) to give (S)-allyl2-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexyl)amino)acetate(7, 0.51 g, 54% yield for two steps) as an oil. LC/MS (ESI⁻) m/z=552.2(M+H)⁺.

Preparation of Compound 8

Hunig's base (0.19 mL, 1.11 mmol) and di-tert-butyl dicarbonate (0.22 g,1.02 mmol) were added to a solution of (S)-allyl2-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexyl)amino)acetate (7, 0.51 g,0.92 mmol) in DCM (4 mL) under argon. The mixture was stirred for 4 h,at which time water was added and the product was extracted into DCM(3×). The combined extracts were dried over anhydrous magnesium sulfate,filtered, and concentrated in vacuo to give an oil. The oil was purifiedby silica gel chromatography (0 to 100% EtOAc/heptane gradient) to give(S)-allyl2-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexyl)(tert-butoxycarbonyl)amino)acetate(7, 0.52 g, 86% yield) as an oil. LC/MS (ESI⁻) m/z=674.2 (M+Na)⁺.

Preparation of Compound 9

Tetrakis(triphenylphosphine)palladium(0) (91 mg, 0.078 mmol) andphenylsilane (0.19 mL, 1.57 mmol) were added to a degassed solution of(S)-allyl2-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexyl)(tert-butoxycarbonyl)amino)acetate(8, 0.52 g, 0.78 mmol) in DCM (5 mL). The mixture was sparged with argonfor 3 min and then stirred at RT for 2 h, at which time water was addedand the product was extracted into DCM (2×). The combined extracts weredried over anhydrous magnesium sulfate, filtered, and concentrated invacuo to give an oil. The oil was purified by silica gel chromatography(0 to 10% MeOH/DCM gradient) to give product as a brown solid. Thissolid was then dissolved in DCM (10 mL) and then a colored impurity wasremoved by stirring with siliabond DMT silica gel (300 wt %) for 1 h at30° C. The silica gel was filtered off using celite and the filtrate wasconcentrated in vacuo to give(S)-2-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexyl)(tert-butoxycarbonyl)amino)aceticacid (9, 0.36 g, 76% yield) as a white solid. LC/MS (ESI⁻) m/z=634.2(M+Na)⁺.

Preparation Entry Intermediate Aldehyde of aldehyde Amine Mass 1¹

Scheme 2

645.3 (M + Na) 2 

Scheme 2

763.3 (M + H) 3²

Scheme 2

948.2 (M + H) ¹reductive amination carried out using NaCNBH₃ in 1:1DCM/MeOH. ²Allyl ester deprotection carried out using Pd(OAc)₂, P(OEt)₃,and dimedone, (Seki, M.; Kondo, K.; Kuroda, T.; Yamanaka, T.; Iwasaki,T., SynLett., 1995, 609-611).

Preparation of Compound 1b

A suspension of commercially available (1a) (6.0 g, 9.82 mmol),paraformaldehyde (2.95 g, 29.5 mmol), 4-methylbenzene sulfonic acid,monohydrate (0.093 g, 0.491 mmol) in toluene (150 mL) was heated to 75°C. for 30 min. The solution was cooled to 20° C. then extracted with 5%NaHCO₃ (2×25 mL). The organic was dried over MgSO₄, filtered, thenconcentrated onto dry silica (10 g) under reduced pressure. The productswere then purified by silica gel chromatography (220 g) eluting productswith a gradient of 0 to 40% ethyl acetate/heptane to afford(S)-(9H-fluoren-9-yl)methyl5-oxo-4-β-oxo-3-(tritylamino)propyl)oxazolidine-3-carboxylate (1b, 5.95g, 97% yield) as white solid. m/z (APCI, pos. ion) 645.2 (M+Na).

Preparation of Compound 1c

To a stirring solution of (1b) (5.95 g, 9.56 mmol) and triethylsilane(5.56 g, 47.8 mmol) in CHCl₃ (30 mL) at 20° C. was added trifluoroaceticacid (30 mL). The solution was stirred for 18 hrs at 40° C. The solventswere then removed under reduced pressure and the residue azeotroped withglacial AcOH (3×100 mL) to afford (S)-(9H-fluoren-9-yl)methyl5-oxo-4-β-oxo-3-(tritylamino)propyl)oxazolidine-3-carboxylate (1c) aswhite solid that was used on the next step without further purification.m/z (APCI, pos. ion) 383.1 (M+1).

Preparation of Compound 1d

To a stirring suspension (1c) (3.6 g, 9.41 mmol), triphenylmethanol(4.90 g, 18.83 mmol), acetic anhydride (1.922 g, 18.83 mmol) in glacialAcOH (40 mL) was added neat sulfuric acid (0.025 ml, 0.471 mmol) at 20°C. The yellowed mixture was then heated to 45° C. to create a solutionand stirred for 18 hr. The reaction was then partitioned between icewater (200 mL) and EtOAc (200 mL). The organic was dried over MgSO₄,concentrated under reduced pressure, then purified by silica gelchromatography (220 g) eluting products with a gradient of 10 to 30% of3:1 ethyl acetate/ethanol (solvent B) and heptane (solvent A) to afford(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-5-oxo-5-(tritylamino)pentanoicacid (1d, 3.85 g, 6.16 mmol, 65.5% yield) as white foam. m/z (APCI, pos.ion) 647.2 (M+Na).

N-methylation of the peptide backbone was also accomplished directlyaccording to Scanlon et al. Int. J. Pept. Res. Ther. 2008, 14, 381-386or Biron et al. J. Peptide Sci. 2006, 12, 213-219. Briefly, the resinsupported peptide was treated with o-nitrobenzenesulfonyl chloride and2,4,6-collidine followed by methyl p-nitrotriflate and MTBD or withtriphenylphosphine, DIAD and methanol. The intermediateo-nitrobenzenesulfonamide was cleaved by treatment with mercaptoethanoland DBU.

A more detailed description of the preparation of the modified apelinpolypeptides using solid phase peptide synthesis follows.

Experimental Method for Peptide Synthesis

Peptides were prepared on an Intavis MultiPep RSi automatedhigh-throughput peptide synthesizer using N^(α)-Fmoc solid-phase peptidesynthesis (SPPS) methodologies with appropriate orthogonal protectionand resin linker strategies. The peptides were synthesized on 0.1 mmolscale using pre-loaded Fmoc-amino acid resins. The remaining amino acidswere added to the growing peptide chain by stepwise addition usingstandard solid phase methods in a 10 mL fritted reaction vessels. TheN^(α)-Fmoc group was removed from resin bound amino acid by addition of20% piperidine solution in dimethylformamide (DMF, 1×5 min, 1×15 min).Coupling reactions proceeded by preactivation (5 min) of five-foldexcess N^(α)-Fmoc-amino acid, 5-fold excess diisppropylcarbodiimide(DIC), and 5-fold excess 1-Hydroxy-7-azabenzotriazole (HOAt) in peptidesynthesis grade DMF followed by addition to the peptide-resin. The stockconcentrations were 0.5 M amino acid, 1 M DIC, and 0.4 M HOAt. The resinmixture was mixed for 60 min per cycle, drained, and if a double couplecycle was used the coupling cycle was repeated prior to Fmoc removal.Upon complete formation of the protected peptide, the final N^(α)-Fmocprotecting group was removed as described above. N-terminal acylationswere performed as described for amino acid coupling. N-terminalacetylations were performed with 3 mL of 10% acetic anhydride and 0.5 mL1.25 M DIEA in DMF for 1 h. Peptides with lipidation at peptide backboneresidues were prepared by first removing Mtt from the Lys(Mtt) residuewith a 2% TFA/5% Tis in DCM solution (5×10 min), neutralization of theepsilon amine with 10% DIEA in DCM (2×5 min), followed by acylations asdescribed above for amino acid coupling cycles. Peptide-resins werewashed with DMF (4×), DCM (6×), and dried thoroughly in vacuo prior tocleavage.

Experimental Method for Deprotection & Cleavage from Solid Support

The peptides were cleaved from the resin by agitating each peptide-resin5 mL of a solution of trifluoroacetic acid (TFA, 87.5%),triisopropylsilane (Tis, 5%), water (2.5%), Anisole (1%), andthioanisole (1%) for 2 h at room temperature. The crude peptide TFAsolutions were transferred to tarred 50 mL conical tubes by filtrationusing a Biotage Syro cleavage device, the resins washed with 5 mL of theTFA solution, and the filtrates combined. The combined filtrates wereconcentrated in the 50 mL conical polypropylene tubes by vacuumcentrifugation for 2 h (Genevac HT-12), and the crude peptides wererecovered by precipitation with cold anhydrous ethyl ether giving awhite to amber colored precipitate. The 50 mL conical tubes werecentrifuged @ 3000 RPM for 4 min, the ether decanted, the precipitantswashed with additional 50 mL cold ether, centrifuged, decanted, and thendried in vacuo prior to analytical LCMS analysis.

Method for Analytical Analysis of Crude Peptides

Crude peptides were dissolved in 50% ACN in H₂O (0.1% TFA) and thesolutions filtered with 0.45 um syringe filters. Crude analysis sampleswere prepared for LCMS analysis by making 10-fold dilutions in 50% ACNin H₂O (0.1% TFA). Peptides were analyzed by analytical LCMS by elutionfrom a Phenomenex Kinetex C18 column (1.7 μm, 2.1×50 mm). Acetylatedpeptides were characterized using a linear gradient of 5-50% ACN in H₂O(0.1% TFA) over 3 min at a flow rate of 1000 μL/min. PEGylated andlipidated peptides were characterized using a linear gradient of 20-65%ACN in H₂O (0.1% TFA) over 3 min at a flow rate of 1000 μL/min.

General Method for Purification

Peptides were purified by preparative reverse-phase high-pressure liquidchromatography (RP-HPLC) by elution from a YMC Triart C18 column (5 um,30×250 mm). The typical method consisted of a 4 min hold atequilibration conditions followed by a linear gradient of 0.333%increase of acetonitrile in H₂O (0.1% TFA) over 56 min @ 40 mL/min. Thehigh purity fractions corresponding to correct products were collected,combined, concentrated in vacuo, and lyophilized to afford an amorphoussolid.

General Method for Characterization

Purified products were analyzed for purity and MW verification on anAgilent 1290 LC-MS system by elution from a Phenomenex Max-RP column(2.5 μm, 2.0×50 mm). Acetylated peptides were characterized using alinear gradient of 5-50% ACN in H₂O (0.1% TFA) over 10 min at a flowrate of 700 μL/min. PEGylated and lipidated peptides were characterizedusing a linear gradient of 20-65% ACN in H₂O (0.1% TFA) over 10 min at aflow rate of 700 μL/min. Peptides were quantified by ChemiluminescentNitrogen Detection (CLND). Peptides with ≥95% purity at 214 nM and withtheoretical mass observed were registered in the database and submittedfor additional in vitro and in vivo characterization as described in thefollowing examples.

Example 2. Preparation of Lipidated Apelin Polypeptides

Lipidation of the modified apelin polypeptides is one strategy forextending the half-life of the modified polypeptides in vivo. A genericdescription of the strategy follows:

A represents a fatty acyl group, where R=carboxylic acid, amine,hydroxyl, ester, alkene, alkyne, or methyl. B represents γGlu(α-carboxy-4-aminobutyric acid; AC4Abu) for acidity & solubility. C is ashort PEG group, such as [2-(2-Amino-ethoxy)-ethoxy]-acetic acid (Aeea)for flexibility & solubility. X=point of covalent attachment to theapelin polypeptide. As independent or joint constituents B and Ccomprise the conjugation linker. The conjugation linker is not criticalto achieve half-life protraction and does not contribute to theintrinsic APJ agonist activity of the apelin polypeptide but serves aspatial, functional and/or structural role between the fatty acylhalf-life extension moiety and the apelin polypeptide. In this regardthe conjugation linker may be absent or can be substituted with anyother moiety that accomplishes the covalent attachment of the fatty acylhalf-life extension group.

Fatty acids are covalently attached to the N-terminus, or amino acidscorresponding to positions 70 or 71 in the apelin preprotein (SEQ ID NO:2), or other sites in the peptide. The fatty acyl group can be attacheddirectly to the polypeptide or with a spacer such as: AEEA (small PEGgroup), AEEA-AEEA, γGlu-AEEA, γGlu-AEEA-AEEA, γGlu, or other amino acid.The alkyl chain of the fatty acid is typically composed of 2 to 24methylene units.

Various modified apelin polypeptides were conjugated to severaldifferent fatty acyl groups according to this strategy and the resultinglipidated polypeptides were tested for APJ receptor agonist activity asdescribed in Examples 3 and 10. It should be noted that the apelinpolypeptides in the sequences below and described herein, withoutconjugation (e.g. without conjugation to a fatty acyl group) may alsoserve as APJ agonists.

Example 3. APJ Agonist Activity of Modified Apelin Polypeptides

Greater than 800 modified peptides were used in structure-activityrelationship assays to optimize for potency and metabolic stabilityrelative to the endogenous ligand of the APJ receptor, pyr apelin-13(SEQ ID NO: 6). Compounds were screened up to 10 μM along with pyrapelin-13, which was used as a positive control. Compounds that showedfunctional activity in all the SAR assays were deemed positive andre-screened. The iterative process was continued and peptides werefurther modified for optimization.

Two different assays, which are described below, were employed to assessthe APJ agonist activity of the modified apelin polypeptides. The cAMPassay involves measuring changes in intracellular cAMP as a result ofactivation of the APJ receptor. Activation of the APJ receptor byendogenous ligand pyr-apelin results in a decrease in intracellularcAMP. The second assay, which is the GTPγS assay, measures the couplingof G protein to the APJ receptor when bound to an agonist. Thus, extentof decrease in cAMP levels and efficacy of G protein bound to agonistreceptor complex is used to assess the extent of agonist activity of themodified apelin polypeptides.

cAMP assay

Stable CHO cell lines expressing the human or rat APJ receptor wereincubated with stimulation buffer containing HANK's buffer, forskolinand 0.5 mM IBMX in the absence or presence of various concentrations ofpeptides at 37° C. for 45 min. The cAMP level was determined using acyclic AMP kit (Cisbio cAMP kit) according to the manufacturer'sinstructions.

[³⁵S] GTPγS assay

To assay for GTPγS bound to receptor, membranes were prepared fromstable cell lines expressing the human or rat APJ receptor and thereceptor bound to the non-hydrolyzable GTP (GTPγS) was used to determinethe efficacy/potency of the modified peptides. The optimal experimentalconditions for the concentrations of GDP, MgCl₂ and NaCl in the assaybuffer were initially determined. The membranes were incubated withmodified peptides in the assay buffer. The reaction was initiated byaddition of [³⁵S] GTPγS in the absence or presence of peptides andincubated at room temperature for 90 min. Non-specific binding wasdetermined in the presence of excess cold GTPγS and was always less than0.2% of total binding. Bound [³⁵S] GTPγS was separated from freeradiolabel by filtration. The filter bound radioactivity was determinedby liquid scintillation counting.

GTPγS assay results for various modified apelin polypeptides are shownin Table 11 below. EC50 values are provided for each modifiedpolypeptide for activation of the human and rat APJ receptors

TABLE 11 APJ Agonist Activity of Modified Apelin Polypeptides EC50 EC50Human Rat SEQ APJ APJ ID Receptor Receptor NO: SEQUENCE (μM) (μM) 6{Hydrogen}[PE] .0046608 .023416 [pyra- RPRLSHKGPMPF{COOH} pelin] 16Acetyl-[hArg][r]Q .000375 .00072863 [hArg]P[r][NMeLeu]SHKG[Oic][pI-Phe]P[D- Bip]{COOH} 17 Acetyl-RQRP[hArg][Cha] .00017065.001588 SHKG[Oic][Nle]P[4-Cl-F]{COOH} 18 Acetyl-[r][hArg]Q[hArg]P[r].00046783 .000523 [NMeLeu]SHKG[Oic][pI-Phe]P[D- Bip]{COOH} 19Acetyl-[hArg][hArg][q][hArg] .00053333 .00075733P[r][NMeLeu]SHKG[Oic][pI-Phe] P[D-Bip]{COOH} 20Acetyl-[hArg][hArg]Q[r]P[r] .000189 .000706[NMeLeu]SHKG[Oic][pI-Phe]P[D- Bip]{COOH} 21 Acetyl-[hArg][hArg]Q[hArg].00057 .0018 [Oic][r][NMeLeu]SHKG[Oic][pI- Phe]P[D-Bip]{COOH} 22Acetyl-[hArg][hArg]Q[r] .0015167 .0045233 P[NMeArg][Cha]SHKG[Oic][pI-Phe]P [D-Bip]{COOH} 23 Acetyl-[hArg]Q[r]P[NMeArg]0.000384 0.0322 [Cha]SHKG[Oic][Nle]P[4-Cl-F] {COOH} 24Acetyl-[hArg]Q[r]P[NMeArg] 0.00787 0.134 [Cha]SHKG[Oic][Nle]P[D-4ClF]{COOH} 25 Acetyl-[hArg]Q[r]P[NMeArg] 0.001735 0.053675[Cha]SHKG[Oic][Nle][D-1Nal] {COOH} 29 Acetyl-OF[hArg][hArg][BLeu].0001155 .00030133 [hArg]P[hArg][Cha]SHKG[Oic] [Nle]P[4-Cl-F]{COOH} 30Acetyl-OF[hArg][hArg]Q .000166 .00045633 [hArg]P[hArg][BLeu]SHKG[Oic][Nle]P[4-Cl-F]{COOH} 31 {Acetyl}OF[hArg][hArg]Q 0.0001155 0.000703[hArg]P[hArg][Cha][NhSerG]HKG [Oic][Nle]P[4-Cl-F]{COOH} 32{Acetyl}OF[hArg][hArg]Q 0.00025033 0.001514 [hArg]P[hArg][Cha]SH[NLysG]G[Oic][Nle]P[4-Cl-F]{COOH} 33 {Acetyl}OF[hArg][hArg]Q 0.0000985330.00031767 [hArg][aMePro][hArg][Cha]SHKG [Oic][Nle]P[4-Cl-F]{COOH} 34{Acetyl}OF[hArg][hArg]Q[hArg] 0.000051933 0.00019267P[hArg][aMeLeu]SHKG[Oic] [Nle]P[4-Cl-F]{COOH} 35 {Acetyl}OF[hArg][hArg]0.000086117 0.00018455 Q[hArg]P[hArg][Cha][aMeS]HKG[Oic][Nle]P[4-Cl-F]{COOH} 36 {Acetyl}OF[hArg][hArg]Q 0.000354670.0019433 [hArg]P[hArg][Cha]SH[aMeOrn]G [Oic][Nle]P[4-Cl-F]{COOH} 37{Acetyl}OF[hArg][hArg]Q[hArg] 0.0000672 0.00013573P[hArg][Cha]SHKG[Oic][Nle] [aMePro][4-Cl-F]{COOH} 38{Acetyl}OF[hArg][hArg]Q[rhArg] 0.000086933 0.0013 P[hArg][Cha]SHKG[Oic][Nle]P [4-Cl-F]{COOH} 39 {Acetyl}OF[hArg][hArg]Q[hArg] 0.000252670.00032333 P[rhArg][Cha]SHKG[Oic] [Nle]P [4-Cl-F]{COOH} 40{Acetyl}OF[hArg][hArg]Q[hArg] 0.00023367 0.00145P[hArg][Cha]S[rHis]KG[Oic] [Nle]P[4-Cl-F]{COOH} 41{Acetyl}OF[hArg][hArg]Q[hArg] 0.00015097 0.000817P[hArg][Cha]SH[rLys]G[Oic] [Nle]P[4-Cl-F]{COOH} 42{Acetyl}OF[hArg][hArg] 0.000044933 0.000247Q[hArg]P[hArg][rCha]SHKG[Oic] [Nle]P[4-Cl-F]{COOH} 43{Acetyl}OF[hArg][hArg]Q[hArg] 0.000393 0.0018267P[hArg][Cha][rSer]HKG[Oic] [Nle]P[4-Cl-F]{COOH} 44{Acetyl}OF[hArg][hArg] 0.00099667 0.00379 Q[hArg]P[hArg][Cha][DrSer]HKG[Oic][Nle]P[4-Cl-F]{COOH} 46 {Acetyl-NH}OF[hArg][hArg] .0003895 .0010408QRP[hArg][Cha]SHKGP[pI-Phe]P [D-Bip]{COOH} 47 {Acetyl-NH}OF[hArg][hArg].00041633 .00063017 QRP[hArg][NMeLeu]SHKG[Oic] [pI-Phe]P[D-Bip]{COOH} 48{Acetyl-NH}OF[hArg][hArg]QRP 0.00028683 0.0004865[hArg][Cha]SHKGP[Nle][4-Cl-F] {COOH} 49 {Acetyl-NH}OF[hArg][hArg]0.00015867 0.000291 QRP[hArg][Cha]SHKGP[Nle][2- Nal]{COOH} 50{Acetyl-NH}rQ[hArg]Pr[NMeLeu] 0.0014333 0.0016767SHKG[Oic][pI-Phe]P[D-Bip] {COOH} 51 {Acetyl-NH}RQRP[hArg] 0.000370.0012615 [NMeLeu]SHKG[Oic][pI-Phe]P[D- Bip]{COOH} 52{Acetyl-NH}rQRP[hArg][NMeLeu] 0.000623 0.001144 SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 53 {Acetyl-NH}Q[hArg]Pr[NMeLeu] 0.00518 0.005984SHKG[Oic][pI-Phe]P[D-Bip] {COOH} 54 {Acetyl-NH}QrP[NMeArg] 0.0301220.31175 [Cha]SHKG[Oic][Nle][D-1Nal] {COOH} 55{Acetyl-NH}q[hArg]P[NMeArg] 0.031578 0.14514 [Cha]SHKG[Oic][Nle]P[D-4ClF]{COOH} 56 {Acetyl-NH}[hArg][hArg]Q 0.0001755 0.00091483[hArg]P[NMeArg][Cha]SHKG[Oic] [Nle]P[4-Cl-F]{COOH} 57{Acetyl-NH}OF[hArg][hArg] 0.00041167 0.00078667Q[hArg]P[NMeArg][Cha]SHKG [Oic][Nle][4-Cl-F]{COOH} 58{Acetyl-NH}OF[hArg][hArg] 0.0011692 0.005315 Q[hArg][Oic]r[Cha]SHKG[Oic][Nle][4-Cl-F]{COOH} 59 {Acetyl-NH}OF[hArg][hArg] 0.0020833 0.0045733Q[hArg]Pr[NMeLeu]SHKG[Oic] [Nle][4-Cl-F]{COOH} 96{H2}[PE]RP[hArg][Cha][Pra] .0020206 .014617(NPeg9)HKG[Oic][Nle]P[4-Cl-F] {COOH} 97 [MerPr](20K mPEGacetamide).00020267 .000253 KFRRQRP[hArg][Cha]SHKG [Oic][Nle]P[4-Cl-F]{COOH} 100{H2}[MerPr](20K mPEGacetamide) 0.00806 0.0454 RQRP[hArg][Cha]SHKG[Oic][Nle]P[4-Cl-F]{COOH} 101 {H2}[MerPr](20K mPEGacetamide) 0.0466670.1496 QRP[hArg][Cha]SHKG [Oic][Nle]P[4-Cl-F]{COOH} 102{Npeg11}KFRRQRP[hArg][Cha] .000083033 .00017967 SHKG[Oic][Nle]P[4-Cl-F]{COOH} 103 {H2}[3TP](20K-mPEGReg)[hArg] .034233 .0522rQ[hArg]Pr[NMeLeu]SHKG [Oic][pI-Phe]P[D-Bip]{COOH} 104{H2}[3TP](20K-mPEGReg)[hArg] .018867 .045033 RQ[hArg]PR[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 105 {Acetyl}[Atz(20K- .000187 .000285mPEG)]KFRRQRP [hArg][Cha]SHKG[Oic] [Nle]P[4-Cl-F]{COOH} 106{Acetyl}[Atz(20K- .0032235 .0473 mPEG)]LRP[hArg] [Cha]SHKG[Oic][Nle]P[4-Cl-F]{COOH} 107 {Acetyl-NH}[Pra](20K- .010547 .010047mPEGReg)OF[hArg] [hArg]QRP[hArg] [NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 126 {TDA}[AC4Abu][Aeea][Aeea] .011 .056167[q][hArg]P[NMeArg][Cha]SHKG [Oic][Nle]P[f]{COOH} 149{TDA}[AC4Abu][Aeea][Aeea] 0.0031867 0.0099867 [hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[f]{COOH} 153 {TDA}[AC4Abu][Aeea][Aeea] 0.0037550.07205 [r]Q[hArg]P[r][NMeLeu]SHKG [Oic][pI-Phe]P[Oic]{COOH} 154{TDA}[AC4Abu][Aeea][Aeea][r] 0.00454 0.04045 Q[hArg]P[r][NMeLeu]SHKG[Oic][pI-Phe]P[D-4ClF]{COOH} 155 {TDA}[AC4Abu][Aeea][Aeea][r] 0.0086250.0496 Q[hArg]P[r][NMeLeu]SHKG [Oic][pI-Phe]P[D-1Nal]{COOH} 165{TDA}[AC4Abu][Aeea][Aeea] .00116 .030267 [hArg]Q[r]P[NMeArg][Cha]SHKG[Oic][Nle][D-1Nal]{COOH} 173 {TDA}[AC4Abu][Aeea][Aeea][r] 0.0016550.01367 Q[hArg]P[r][NMeLeu]SHKG [Oic][pI-Phe][D-Bip]{COOH} 174{TDA}[AC4Abu][Aeea][Aeea] 0.003605 0.01655 [hArg][q][hArg]P[r][NMeLeu]SHKG[Oic][pI-Phe] [D-Bip] {COOH} 175 {TDA}[AC4Abu][Aeea][Aeea] 0.0013250.008545 [hArg]Q[r]P[r][NMeLeu]SHKG [Oic][pI-Phe][D-Bip]{COOH} 180{TDA}[AC4Abu][Aeea][Aeea] 0.01004 0.0273 [hArg][q][hArg]P[r][BLeu]SHKG[Oic][pI-Phe] P[D-Bip]{COOH} 199 {TDA}[AC4Abu][Aeea][Aeea] 0.008480.0324 [hArg][q][hArg]P[r][NMeLeu] SAKG[Oic][pI-Phe]P [D-Bip] {COOH} 201{TDA}[AC4Abu][Aeea][Aeea] 0.0494 0.1825 [hArg][q][hArg]P[r][NMeLeu]S[Tle]KG[Oic][pI-Phe] P[D-Bip]{COOH} 208 {TDA}[AC4Abu][Aeea][Aeea] 0.02890.104 [hArg][q][hArg]P[r][NMeLeu] [bAla]HKG[Oic] [pI-Phe]P[D-Bip]{COOH}211 {TDA}[AC4Abu][Aeea][Aeea] 0.0019033 0.0062867RQRP[r][NMeLeu]SHKG[Oic][pI- Phe]P[D-Bip]{COOH} 212{TDA}[AC4Abu][Aeea][Aeea] .00051533 .0013663 RQRP[hArg][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 213 {TDA}[AC4Abu][Aeea][Aeea][r] .00064667.0011737 QRP[hArg][NMeLeu]SHKG [Oic][pI-Phe]P[D-Bip]{COOH} 214{TDA}[AC4Abu][Aeea][Aeea] 0.0029233 0.0058067RQRP[hArg][Cha]SHKG[Oic][pI- Phe]P[D-Bip]{COOH} 215{TDA}[AC4Abu][Aeea][Aeea][r] 0.0084233 0.0214QRP[r][Cha]SHKG[Oic][pI-Phe] P[D-Bip]{COOH} 216{TDA}[AC4Abu][Aeea][Aeea] 0.01076 0.0225 RQRP[r][Cha]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 217 {TDA}[AC4Abu][Aeea][Aeea][r] 0.0046667 0.00883QRP[hArg][Cha]SHKG[Oic][pI- Phe]P[D-Bip]{COOH} 218{TDA}[AC4Abu][Aeea][Aeea][r] .00084133 .014933 Q[hArg]P[r][NMeLeu]SHKG[Oic][Nle]P[4-Cl-F]{COOH} 219 {TDA}[AC4Abu][Aeea][Aeea][r] 0.0101430.045 Q[hArg]P[r][NMeLeu]SHKG [Oic][pI-Phe]P[4-Cl-F] {COOH} 220{TDA}[AC4Abu][Aeea][Aeea][r] 0.0027233 0.0276 Q[hArg]P[r][NMeLeu]SHKG[Oic][Nle]P[D-Bip]{COOH} 221 {TDA}[AC4Abu][Aeea][Aeea][r] .00019567.00193 Q[hArg]P[r][Cha]SHKG[Oic] [Nle]P[4-Cl-F]{COOH} 222{TDA}[AC4Abu][Aeea][Aeea] .0012267 .0068667 QRP[hArg][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 223 {TDA}[AC4Abu][Aeea][Aeea] 0.0112570.052767 QRP[hArg][Cha]SHKG[Oic][pI- Phe]P[D-Bip]{COOH} 224{TDA}[AC4Abu][Aeea][Aeea] 0.028167 0.1025 QRP[r][Cha]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 225 {TDA}[AC4Abu][Aeea][Aeea] 0.0030233 0.0479Q[hArg]P[r][NMeLeu]SHKG [Oic][Nle]P[4-Cl-F]{COOH} 226{TDA}[AC4Abu][Aeea][Aeea] 0.020767 0.091 Q[hArg]P[r][NMeLeu]SHKG[Oic][pI-Phe]P[4-Cl-F]{COOH} 227 {TDA}[AC4Abu][Aeea][Aeea] 0.008770.097533 Q[hArg]P[r][NMeLeu]SHKG [Oic][Nle]P[D-Bip]{COOH} 228{TDA}[AC4Abu][Aeea][Aeea] .00048433 .00791Q[hArg]P[r][Cha]SHKG[Oic][Nle] P[4-Cl-F]{COOH} 229{TDA}[AC4Abu][Aeea][Aeea] .00023533 .00329 [hArg]Q[r]P[NMeArg][Cha]SHKG[Oic][Nle]P[4-Cl-F]{COOH} 230 {TDA}[AC4Abu][Aeea][Aeea] .0013667 .013833[hArg]Q[r]P[NMeArg][Cha]SHKG [Oic][Nle]P[D-4ClF]{COOH} 234{HDA}[AC4Abu][Aeea][Aeea] .0060533 .0125 Q[r]P[NMeArg][Cha]SHKG[Oic][Nle][Pip][4-Cl-F]{COOH} 237 {HDA}[AC4Abu][Aeea][Aeea][q] .0049633.0048933 [hArg]P[NMeArg][Cha]SHKG [Oic][Nle]P[D-4ClF]{COOH} 238{HDA}[AC4Abu][Aeea][Aeea] .00668 .0285 Q[r]P[NMeArg][Cha]SHKG[Oic][Nle]P[Tic]{COOH} 239 {HDA}[AC4Abu][Aeea][Aeea] .00145 .0086067[hArg]Q[r]P[NMeArg][Cha]SHKG [Oic][Nle][Pip][4-Cl-F] {COOH} 242{HDA}[AC4Abu][Aeea][Aeea] .00219 .00227 [hArg][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P [D-4ClF] {COOH} 243 {HDA}[AC4Abu][Aeea][Aeea] 0.00119220.010252 [hArg]Q[r]P[NMeArg][Cha]SHKG [Oic][Nle]P[Tic]{COOH} 244{TDA}[AC4Abu][Aeea][Aeea] 0.010297 0.28067 Q[r]P[NMeArg][Cha]SHKG[Oic][Nle]P[Tic]{COOH} 257 {TDA}[AC4Abu][Aeea][Aeea]OF .0014853 .003135[hArg][hArg]QRP[hArg][NMeLeu] SHKG[Oic][Nle][2Nal]{COOH} 258{PDA}[AC4Abu][Aeea][Aeea]OF .0017533 .0025717[hArg][hArg]QRP[hArg][NMeLeu] SHKG[Oic][Nle][2Nal]{COOH} 259{HDA}[AC4Abu][Aeea][Aeea]OF .0025917 .0020917[hArg][hArg]QRP[hArg][NMeLeu] SHKG[Oic][Nle][2Nal]{COOH} 260{ODDA}[AC4Abu][Aeea][Aeea]OF .048533 .07495[hArg][hArg]QRP[hArg][NMeLeu] SHKG[Oic][Nle][2Nal]{COOH} 261{TDA}[AC4Abu][Aeea][Aeea] .0047383 .009375 [hArg]rQ[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 262 {PDA}[AC4Abu][Aeea][Aeea] .0044233.0067133 [hArg]rQ[hArg]Pr[NMeLeu]SHKG [Oic][pI-Phe]P[D-Bip]{COOH} 263{HDA}[AC4Abu][Aeea][Aeea] .0039922 .00591 [hArg]rQ[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 264 {ODDA}[AC4Abu][Aeea][Aeea] .1449 .19333[hArg]rQ[hArg]Pr[NMeLeu] SHKG[Oic][pI-Phe] P[D-Bip]{COOH} 265{TDA}[AC4Abu][Aeea][Aeea] 0.0012 0.01447 q[hArg]Pr[Cha]SHKG[Oic][Nle]P[D-4ClF]{COOH} 266 {TDA}[AC4Abu][Aeea][Aeea] 0.00581 0.03325QrPr[Cha]SHKG[Oic][Nle]P [D-4ClF]{COOH} 267 {TDA}[AC4Abu][Aeea][Aeea].00075333 .0176 QrP[NMeArg][Cha]SHKG[Oic] [Nle]P[4-Cl-F]{COOH} 269{TDA}[AC4Abu][Aeea][Aeea] 0.073 0.0523 q[hArg]Pr[Cha]SHKG[Oic][Nle]P[D-Tic]{COOH} 270 {TDA}[AC4Abu][Aeea][Aeea]q 0.001755 0.001489[hArg]Pr[Cha]SHKG[Oic][Nle] [Aib][4-Cl-F]{COOH} 271{TDA}[AC4Abu][Aeea][Aeea]q 0.0118 0.007875 [hArg]Pr[Cha]SHKG[Oic][Nle][Deg][4-Cl-F]{COOH} 272 {TDA}[AC4Abu][Aeea][Aeea]q 0.01675 0.01155[hArg]Pr[Cha]SHKG[Oic][Tle]P [4-Cl-F]{COOH} 273{TDA}[AC4Abu][Aeea][Aeea]q 0.001665 0.00509 [hArg]Pr[Cha]S[Aib]KG[Oic][Nle]P[4-Cl-F]{COOH} 274 {TDA}[AC4Abu][Aeea][Aeea]q 0.008045 0.0462[hArg]Pr[BLeu]SHKG[Oic][Nle]P [4-Cl-F]{COOH} 275{TDA}[AC4Abu][Aeea][Aeea]q 0.0034275 0.019342 [hArg]P[NMeArg][BLeu]SHKG[Oic][Nle]P[4-Cl-F]{COOH} 277 {TDA}[AC4Abu][Aeea][Aeea]l 0.016145 0.0209[hArg]Pr[Cha]SHKG[Oic][Nle]P [4-Cl-F]{COOH} 278{TDA}[AC4Abu][Aeea][Aeea] 0.01006 0.0145 [BLeu][hArg]Pr[Cha]SHKG[Oic][Nle]P[4-Cl-F]{COOH} 279 {TDA}[AC4Abu][Aeea][Aeea] 0.00329 0.019275Q[NMeArg]Pr[Cha]SHKG[Oic] [Nle]P[4-Cl-F]{COOH} 280{TDA}[AC4Abu][Aeea][Aeea] 0.002045 0.00447 q[NMeArg]Pr[Cha]SHKG[Oic][Nle]P[4-Cl-F]{COOH} 281 {TDA}[AC4Abu][Aeea][Aeea]q 0.0148 0.08735[hArg]Pr[BLeu]SHKG[Oic][Nle]P [D-4ClF]{COOH} 282{TDA}[AC4Abu][Aeea][Aeea] 0.00579 0.0133 q[hArg]Pr[Cha]SHKG[Oic][Nle][4-Cl-F]{COOH} 283 {TDA}[AC4Abu][Aeea][Aeea]q 0.00974 0.0156[hArg]Pr[Cha]SHKG[Oic][Nle] [D-4ClF]{COOH} 284{TDA}[AC4Abu][Aeea][Aeea] >4.17 >4.17 QrP[NMeArg][Cha]SHKG[Oic][Nle]P[D-4ClF]{COOH} 285 {TDA}[AC4Abu][Aeea][Aeea] .0082233 .122QrP[NMeArg][Cha]SHKG[Oic] [Nle][D-1Nal]{COOH} 669OF[NMeArg][hArg]Q[hArg]P 0.0053033 0.0055933 [hArg][Cha]SHKG[Oic][Nle]P[4-Cl-F] 670 OF[hArg][NMeArg]Q[hArg]P 0.00013127 0.000254[hArg][Cha]SHKG[Oic][Nle] P[4-Cl-F] 671 OF[hArg][hArg]Q[NMeArg]P0.00016167 0.00038733 [hArg][Cha]SHKG[Oic][Nle] P[4-Cl-F] 672OF[hArg][hArg]Q[NMeArg]P 0.00044483 0.00084467[NMeArg][Cha]SHKG[Oic][Nle]P [4-Cl-F] 673 OF[hArg][NMeArg]Q[NMeArg]0.0013433 0.0013433 P[NMeArg][Cha]SHKG[Oic] [Nle]P[4-Cl-F] 674[hArg]Q[hArg]P[NMeArg][Cha] 0.00015215 0.000304 SHKG[Oic][Nle]P[4-Cl-F]675 Q[hArg]P[NMeArg][Cha] 0.00015933 0.000189 SHKG[Oic][Nle]P[4-Cl-F]676 OF[hArg][hArg]Q[hArg][Sar] 0.00076217 0.00084033[hArg][Cha]SHKG[Oic][Nle] P[4-Cl-F] 677 OF[hArg][hArg]Q[hArg] 0.00350030.0034803 [Aib][hArg] [Cha]SHKG[Oic][Nle]P[4-Cl-F] 678OF[hArg][hArg]Q[hArg]P[hArg] 0.000664 0.0021133[Cha][Sar]HKG[Oic][Nle]P[4-Cl-F] 679 OF[hArg][hArg]Q[hArg]P[hArg]0.0036267 0.033 [Cha]S[NMeVal]KG[Oic][Nle]P [4-Cl-F] 680OF[hArg][hArg]Q[hArg]P[hArg] 0.0001161 0.00040067[Cha]SHK[Sar][Oic][Nle]P[4-Cl-F] 681 OF[hArg][hArg]Q[hArg]P[hArg]0.00072267 0.0013033 [Cha]SHK[Aib][Oic][Nle]P[4-Cl-F] 682OF[hArg][hArg]Q[hArg]P[hArg] 0.0015347 0.00798[Cha]SHKG[Aib][Nle]P[4-Cl-F] 683 OF[hArg][hArg]Q[hArg]P[hArg] 0.000378670.000926 [Cha]SHKG[Sar][Nle]P[4-Cl-F] 685 OF[hArg][hArg]Q[hArg]P0.0000946 0.00016767 [hArg][Cha]SHKG[Oic][Nle][Aib] [4-Cl-F] 686O[BhPhe][hArg][hArg]Q[hArg] 0.00010907 0.00018767P[hArg][Cha]SHKG[Oic][Nle]P [4-Cl-F] 687 O[BPhe][hArg][hArg]Q[hArg]P0.0000918 0.00017533 [hArg][Cha]SHKG[Oic][Nle]P [4-Cl-F] 689OF[hArg][BhArg]Q[hArg]P[hArg] 0.0001137 0.00034933[Cha]SHKG[Oic][Nle]P[4-Cl-F] 690 OF[hArg][hArg][BhGln][hArg] 0.0001110.00018333 P[hArg][Cha]SHKG[Oic][Nle]P [4-Cl-F] 691OF[hArg][hArg][BhAsn][hArg] 0.00045267 0.000424P[hArg][Cha]SHKG[Oic][Nle]P [4-Cl-F] 692 OF[hArg][hArg][BhLeu][hArg]0.00048533 0.00050333 P[hArg][Cha]SHKG[Oic][Nle]P [4-Cl-F] 693OF[hArg][hArg]Q[BhArg]P[hArg] 0.00022933 0.00050467[Cha]SHKG[Oic][Nle]P[4-Cl-F] 694 OF[hArg][hArg]Q[hArg][BhPro] 0.000457670.00060167 [hArg][Cha]SHKG[Oic][Nle]P [4-Cl-F] 695OF[hArg][hArg]Q[hArg][Pip] 0.00031333 0.000384 [hArg][Cha]SHKG[Oic][Nle]P[4-Cl-F] 696 OF[hArg][hArg]Q[hArg]P[BhArg] 0.0002610.00029967 [Cha]SHKG[Oic][Nle]P[4-Cl-F] 697 OF[hArg][hArg]Q[hArg]P[hArg]0.000225 0.000495 [BhLeu]SHKG[Oic][Nle]P[4-Cl-F] 698OF[hArg][hArg]Q[hArg]P[hArg] 0.00053867 0.00073367[Cha][BhSer]HKG[Oic][Nle]P [4-Cl-F] 699 OF[hArg][hArg]Q[hArg]P[hArg]0.00044033 0.000603 [Cha][bAla]HKG[Oic][Nle]P [4-Cl-F] 700OF[hArg][hArg]Q[hArg]P[hArg] 0.00094933 0.0031133[Cha]S[bAla]KG[Oic][Nle]P [4-Cl-F] 701 OF[hArg][hArg]Q[hArg]P[hArg]0.000316 0.00087933 [Cha]SH[BhLys]G[Oic][Nle]P [4-Cl-F] 702OF[hArg][hArg]Q[hArg]P[hArg] 0.00033 0.0014867 [Cha]SH[BLys]G[Oic][Nle]P[4-Cl-F] 703 OF[hArg][hArg]Q[hArg]P[hArg] 0.00126 0.0026667[Cha]SH[bAla]G[Oic][Nle]P [4-Cl-F] 704 OF[hArg][hArg]Q[hArg]P[hArg]0.00096333 0.0028567 [Cha]SHK[bAla][Oic][Nle]P [4-Cl-F] 705OF[hArg][hArg]Q[hArg]P[hArg] 0.0034833 0.011063[Cha]SHKG[bAla][Nle]P[4-Cl-F] 706 OF[hArg][hArg]Q[hArg]P[hArg] 0.00198670.0043867 [Cha]SHKG[BhPro][Nle]P[4-Cl-F] 707OF[hArg][hArg]Q[hArg]P[hArg] 0.000294 0.00047133[Cha]SHKG[Pip][Nle]P[4-Cl-F] 708 OF[hArg][hArg]Q[hArg]P[hArg] 0.007460.0234 [Cha]SHKG[Oic][bAla]P[4-Cl-F] 709 OF[hArg][hArg]Q[hArg]P[hArg]0.000555 0.00086167 [Cha]SHKG[Oic][Nle][BhPro] [4-Cl-F] 710OF[hArg][hArg]Q[hArg]P[hArg] 0.000785 0.00179 [Cha]SHKG[Oic][Nle][bAla][4-Cl-F] 711 OF[hArg][hArg]Q[hArg]P[hArg] 0.00022867 0.000351[Cha]SHKG[Oic][Nle][Pip][4-Cl-F] 712 OF[hArg][hArg]Q[hArg]P[hArg]0.00065433 0.000821 [Cha]SHKG[Oic][Nle][D-1Nal] 713OF[hArg][hArg]Q[hArg]P[hArg] 0.00154 0.001137[Cha]SHKG[Oic][Nle][D-2Nal] 714 OF[hArg][hArg]Q[hArg]P[hArg] 0.00016820.00027138 [Cha]SHKG[Oic][Nle]P[4-Cl-F]

Example 4. Apelin Polypeptides Improve Cardiac Function

To assess the hemodynamic cardiac function of the peptides, aLangendorff isolated heart system was used. The isolated heart from ratprovides a broad spectrum of measurements that can include biochemical,metabolic, morphological and physiological indices. Although the systemlacks neurohormonal influences, neural regulation and high coronaryflows, the advantages of the system include the ability to study directcardiac effects without systemic circulation and a host of peripheralinteractions. This method can yield excellent dose-responserelationships and provides critical insight into the pharmacological andphysiological properties of the molecules of interest.

Animals were anesthetized through administration of 50-100 mg/kg dose ofsodium pentobarbital via the intraperitoneal route. The heart wasexcised and gently cradled between fingers to avoid injury followed bylifting slightly before incising the aorta, vena cava and pulmonaryvessels. Immediately after excision of the heart with aorta, the heartwas mounted onto the cannula in the Langendorff apparatus via the aorta.The heart was perfused with modified oxygenated Krebs-Henseleit bufferat pH7.5 and equilibrated with 95% O₂/5% CO₂ at 37° C. The perfusion wasperformed at a constant flow of 10 ml/min and paced at 300 bpm. Thepressure was measured through a pressure sensing balloon catheterinserted in the left ventricular cavity. The intrinsic inotropy (forceof muscle contraction) and lusitropy (cardiac relaxation) effects of theheart were assessed by dp/dt_(max) and dp/dt_(min).

The following polypeptides were tested for effects on cardiac functionin the Langendorff isolated heart system.

(SEQ ID NO: 16) Acetyl-[hArg][r]Q[hArg]P[r][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} (SEQ ID NO: 53)Acetyl-Q[hArg]P[r][NMeLeu]SHKG[Oic][pI-Phe]P [D-Bip]{COOH}FIGS. 4A-D and 5A-D illustrate a dose-dependent improvement of bothsystolic and diastolic function in isolated perfused rat hearts withpeptides of SEQ ID NO: 16 and SEQ ID NO: 53, respectively.

Example 5. Apelin Polypeptides Improve Cardiac Function in Rats withHeart Failure

To assess the cardiovascular effects of modified apelin polypeptides,the polypeptides were administered to rats with myocardial infarction(MI)-induced heart failure. Cardiac function was assessed via the MillarPV loop system.

Male Lewis rats at 2-3 month of age were used for the studies. MI wasinduced by ligation of the left anterior descending coronary artery(LAD). Echocardiography was performed at 1 week post-MI for animalenrollment. If ejection fraction (EF) was more than 40% and no infarctwas identified from echocardiography images, these animals were excludedfrom the study. A Millar PV loop catheter was inserted into the rightcommon carotid artery and then advanced to the left ventricle (LV) forcardiovascular hemodynamic assessment. The arterial pressure catheterwas inserted into a femoral artery for peripheral blood pressuremonitoring. Experiments were performed six to seven weeks followinginduction of MI. The following apelin polypeptides were givenintravenously via jugular vein at various doses.

(SEQ ID NO: 6) Pyr-Apelin: {Hydrogen}[PE]RPRLSHKGPMPF{COOH}(SEQ ID NO: 109) {Acetyl-NH}[NPeg11]QRP[hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F]{COOH} (SEQ ID NO: 16)Acetyl-[hArg][r]Q[hArg]P[r][NMeLeu]SHKG[Oic] [pI-Phe]P[D-Bip]{COOH}

Peptides were continuously infused for 30 min at each dose. At the endof the experiment, the infarct size was verified by gross morphology.FIGS. 6-8 show the results of these experiments. The infarct size(38-39% of LV) was very similar among groups. The results demonstratethe dose-dependent effects of pyr-Apelin-13 (SEQ ID NO. 6; FIG. 6A-D)and modified analogues (SEQ ID NOs. 109 and 16; FIGS. 7A-D and 8A-D,respectively) on hemodynamic function in MI rats following continuousinfusion. Contractility (dp/dt max), ejection fraction (EF), meanarterial pressure (MAP), and heart rate (HR) changes over baseline arereported at the end of the 30 min infusion period for each polypeptide.

Example 6. Pharmacokinetic Profile of Modified Apelin Polypeptides

Evaluation of the in vivo pharmacokinetic profile of several pegylatedand lipidated apelin polypeptides in rat was undertaken (FIGS. 9 and10A-B). The apelin polypeptides were administered to Sprague-Dawley ratsthrough subcutaneous (SC) administration and the pharmacokinetic profilefor each polypeptide was subsequently evaluated. A PBS buffer solutioncontaining the apelin peptide was administered to rats via jugular veinto a final dose of 0.5 mg/kg or 5 mg/kg. Blood samples collected over a24 hr period were transferred into collection tubes containing potassiumEDTA and stored on ice until processed. Plasma was obtained from bloodby centrifugation. After transfer into a 96-well container, plasmasamples were stored in a freezer maintained at approximately −80° C.Quantitative analysis of the apelin polypeptide in rat plasma sampleswas achieved using multiple reaction monitor (MRM) mode on a LC-MS/MSsystem.

In vivo pharmacokinetics of a series of 20 kDa PEGylated peptides wasinvestigated in Sprague-Dawley rats. The sequence of the apelinpolypeptide was varied between the peptides tested, whereas the PEGpolymer (20 kDa PEG) was constant. Plasma concentration/exposure can bemodulated by intrinsic metabolic stability of the peptide. As shown inFIG. 9 , high plasma concentrations were achieved with peptide SEQ IDNO: 103 at 1/10^(th) the dose of other 20 kDa PEGylated apelin peptides.

The sequences of the pegylated apelin polypeptides used in thepharmacokinetic study are listed below:

(SEQ ID NO: 105) {Acetyl}[Atz(20-mPEG)]KFRRQRP[hArg][Cha]SHKG[Oic][Nle]P[4-Cl—F]{COOH} (SEQ ID NO: 107){Acetyl-NH}[Pra](20K-mPEG Reg)OF[hArg][hArg]QRP[hArg] [NMeLeu]SHKG [Oic][pI-Phe]P[D-Bip]{COOH} (SEQ ID NO: 108){Acetyl-NH}[Pra](20K-mPEG Reg)OF[hArg][hArg]QRP[hArg] [NMeLeu]SHKG [Oic][pI-Phe]P[pI-Phe]{COOH} (SEQ ID NO: 103){H2}[3TP](20K-mPEG Reg)[hArg]rQ[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH}

In vivo pharmacokinetics of a series of lipidated peptides inSprague-Dawley rats was investigated. In the first set of experiments,the fatty acyl groups (Tridecanoyl, Pentadeconyl, Heptadeconyl, andOctadecandioyl) were varied between the peptides tested, whereas thepeptide sequence remained the same. As shown in FIG. 10A, plasmaconcentration/exposure can be modulated with length & composition of thefatty acyl group. The structure for each of the peptides that were usedin this first set of experiments is depicted below:

(SEQ ID NO: 261) {TDA}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} (SEQ ID NO: 262){PDA}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} (SEQ ID NO: 263){HDA}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} (SEQ ID NO: 264){ODDA}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH}

In the second set of experiments, the sequence of the apelin peptide wasvaried in polypeptide SEQ ID NOs: 237, 213, 212 and 261, whereas thefatty acyl groups differ between polypeptide SEQ ID NOs: 237 and 263 andpolypeptide SEQ ID NOs: 212, 213, and 261. As shown in FIG. 10B, plasmaconcentration/exposure can be modulated by intrinsic metabolic stabilityof the peptide as well as the fatty acyl group.

The structure for each of the peptides that were used in this second setof experiments is depicted below:

(SEQ ID NO: 237) {HDA}[AC4Abu][Aeea][Aeea][q][hArg]P[NMeArg][Cha]SHKG[Oic][Nle]P[D-4ClF]{COOH} (SEQ ID NO: 263){HDA}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} (SEQ ID NO: 212){TDA}[AC4Abu][Aeea][Aeea]RQRP[hArg][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} (SEQ ID NO: 261){TDA}[AC4Abu][Aeea][Aeea][hArg]rQ[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} (SEQ ID NO: 213){TDA}[AC4Abu][Aeea][Aeea][r]QRP[hArg][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH}

Example 7. Effect of Modified Apelin Polypeptides on Mast CellDegranulation

Mast cell degranulation was measured for a number of the modified apelinpolypeptides as follows in order to determine the potential immunogeniceffect of the peptides. Mast cell degranulating peptide (MCDP) andcompound 48/80 were used as positive controls. MCDP was obtained fromAlomone Labs (Israel). Compound 48/80 was obtained from Sigma (SaintLouis, Mich.). Histamine Elisa kit was obtained from NEOGEN (Lexington,Ky.). Tyrode's buffer was obtained from Sigma (#T2397).

Rat Peritoneal Mast Cells

Rat peritoneal fluid was collected in Tyrode's buffer from 8 week oldfemale Sprague Dawley or Lewis rats (Gillespie et al., Histamine releasefrom rat peritoneal mast cells: inhibition by colchicine andpotentiation, 1968; 154-1). Percentage of peritoneal mast cells (5-8%)was determined by Flow Cytometry and immunohistochemistry (IHC).

Human Mast Cells

Mature human mast cells were derived from CD34+peripheral bloodprecursor cells after an 8-12 week differentiation/maturation process invitro. CD34+peripheral blood cells were obtained from AllCells(#MPB016F, donor ID #: PCA1452A) and cultured in the following media for8-12 weeks: SFEM11 Stem Span serum-free media (#09655 StemCellTechnologies), 1× Penicillin (100U/ml) and streptomycin (100 ug/ml)(#15140-122, Life Technologies), 1× Gentamicin/Amphotericin B (Gibco#50-0640), 100 ng/ml SCF (#300-07, PeproTech), IL-3 for first week only(5 ng/ml, #203-IL-050, R&D Systems), IL-9 during week 1-3 (15 ng/ml,#209-IL-050, R&D Systems), IL-6 during week 5-onwards (50 ng/ml,#206-IL-050, R&D Systems) and IL-4 during week 8-onwards (long/ml,#204-IL-050, R&D Systems). Mast cell maturation was confirmed viaexpression of the mast cell markers c-kit and FcεRI via flow cytometry(>75% positive) and histamine release.

Measurement of Mast Cell Degranulation

Mast cells (human or rat) were washed in Tyrode's buffer and seeded on96-well plates (approx. 2000 mast cells/well). A 10 point dilution oftest compounds were prepared in Tyrode's buffer and were incubated(0.005 to 30 μM) with mast cells at 37° C. for 30 min. When testinglipidated molecules, surfactants such as 0.1% Tween 80 from Croda(Edison, N.J.) may be included in Tyrode's buffer to maintain lipidatedmolecule solubility in the assay. A mast cell surfactant control iscompared to a mast cell Tyrode's buffer control to ensure surfactantsare not inducing histamine release in the absence of the lipidatedmolecule. Histamine release was quenched by placing the plates on icefor 5 min. Cells were centrifuged at 350×g at 4° C. for 5 min and thesupernatant was collected. Released histamine was quantified by ELISA(Neogen Corporation) as per the kits' instruction. Briefly, theabsorbance was measured at 650 nm in a microplate reader, following adirect competitive ELISA reaction with test samples compared against astandard curve. Total histamine content of the cells (0.1% Triton X-100)and spontaneous release were also measured to determine the percent ofhistamine release. Percent histamine release was calculated by thefollowing formula:% histamine release=(ng/ml histamine release by test article−ng/mlspontaneous histamine release in Tyrode's buffer)/(ng/ml total histaminecontent of cells lysed in 0.1% Triton X-100−ng/ml spontaneous histaminerelease in Tyrode's buffer)×100.

Table 12 below summarizes the results of the rat mast cell degranulationassay for several modified apelin polypeptides.

TABLE 12 Activity of Apelin Peptides in Inducting Histamine Release fromRat Mast Cells Apelin Polypeptide Cause Histamine EC₅₀ SEQ ID NO.Release? (μM) MCDP Yes 0.52 50 Yes 0.52 51 Yes 0.54 52 Yes 0.55 213 Yes2.74 242 Yes 3.24 212 Yes 4.24 55 Yes 8.17 54 Yes 9.84 237 No *Pyr-Apelin-13; 6 No *

Synthetically modified apelin polypeptides have a range of potencies ininducing in vitro histamine release. SEQ ID NOs: 50-52 are as potent asthe positive control, MCDP, whereas SEQ ID NOs: 237 and 6(pyr-Apelin-13) are inactive at the highest concentrations tested. Itcan be observed from the table that the polypeptide of SEQ ID No. 237does not induce histamine release in the assay.

Example 8. Modified Apelin Polypeptides with Reduced Mast CellDegranulation Capabilities

As shown in Example 7, some modified apelin polypeptides inducehistamine release from rat mast cells, suggesting that thesepolypeptides may have a potential immunogenic effect. To more fullyunderstand the relationship between the structure of the modifiedpolypeptides and the induction of degranulation, and to generateadditional polypeptides with reduced degranulation capability, a furtherstructure-activity relationship (SAR) study was conducted. The modifiedapelin polypeptides in Table 13 below were tested for induction ofhistamine release from rat and human mast cells using the methodsdescribed in Example 7. Compounds that induced less than 30% of thetotal detected histamine content of the cells were deemed to be negativein the assay.

TABLE 13 Mast Cell Degranulation Activity of Modified ApelinPolypeptides Induce Apelin Histamine Poly- Release? peptide Rat HumanSEQ Mast Mast ID NO: Sequence Cells Cells 71 {Acetyl-NH} Q [hArg] No P[NMeArg] [aMeLeu] S H K G P [Nle] P [4-Cl-F]{COOH} 61 {Acetyl-NH} Q[hArg] P No [NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 69{Acetyl-NH} Q R No No [aMePro] [hArg] [aMeLeu] S H K G P [Nle] P[4-Cl-F] {COOH} 70 {Acetyl-NH} Q r P No [hArg] [aMeLeu] S H K G P [Nle]P [4-Cl-F] {COOH} 92 {Acetyl-NH} Q R P [NMeArg] [Cha] S H K G Yes [Oic][Nle] [Aib] [4-Cl-F] {COOH} 68 {Acetyl-NH} Q R No No P [NMeArg] L S H KG P [Nle] P [4-Cl-F] {COOH} 66 {Acetyl-NH} R Q R P No [NMeArg] [Cha] S HK G P [Nle] P [4-Cl-F] {COOH} 67 {Acetyl-NH} R Q R P No [NMeArg][NMeLeu] S H K G P [Nle] P [4-Cl-F] {COOH} 65 {Acetyl-NH} R Q R P[NMeArg] L S H K G P No [Nle] P [4-Cl-F] {COOH} 63 {Acetyl-NH} R Q R P R[Cha] No S H K G P [Nle] P [4-Cl-F] {COOH} 64 {Acetyl-NH} R Q R P R[NMeLeu] S H K G P No [Nle] P [4-Cl-F] {COOH} 62 {Acetyl-NH} R Q R P R LYes S H K G P [Nle] P [D- Bip] {COO} 60 {Acetyl-NH}[hArg]Q[hArg]P Yes[NMeArg][Cha]SHK G [Oic] [Nle] P [4-Cl-F] {COOH} 491 {Butanoyl} [AC4Abu][Aeea] [Aeea] Q R P No No [NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH}116 {H2} [MerPr] (10K mPEG acetamide) Q No [NMehArg] P [NMeArg] [aMeLeu]S H K G P [Nle] P [4-Cl-F] {COOH} 114 {H2} [MerPr] (10K mPEG acetamide)Q R No No [aMePro] [hArg] [aMeLeu] S H K G P [Nle] P [4-Cl-F] {COOH} 119{H2} [MerPr] (10K mPEG No acetamide) Q r P [hArg] [aMeLeu] S H K G P[Nle] P [4-Cl-F] {COOH} 120 {H2} [MerPr] (10K mPEG acetamide) Q R P No[NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl-F] {COOH} 110 {H2}[MerPr] (10K No mPEG acetamide) Q R P R L S H K G P M P F {COOH} 13 {H2}C M P L H S R V P F P {COOH} No 12 {H2} K F R R Q R P R L Yes S H K G PM P {COOH} 496 {HDA} [AC4Abu] [Aeea] Yes [Aeea] Q R P [NMeArg] L S H K GP [Nle] P [4-Cl-F] {COOH} 286 {hexanoyl} [AC4Abu] [Aeea] Yes [Aeea][hArg] r Q [hArg] P r [NMeLeu] S H K G [Oic] [pI-Phe] P [D- Bip] {COOH}290 {ODDA} [AC4Abu] Yes [Aeea] [Aeea] Q R P R L S H K G P M P F {COOH}577 {Palm} [AC4Abu] [Aeea] Yes [Aeea] q R P [NMeArg] [Cha] S H K G [Oic][Nle] [Aib] [BhPhe] {COOH} 578 {Palm} [AC4Abu] [Aeea] Yes [Aeea] q R P[NMeArg] [Cha] S H K G [Oic] [Nle] [BhPhe] {COOH} 600 {Palm} [AC4Abu][Aeea] Yes [Aeea] q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [BhPro][D-4ClF] {COOH} 604 {Palm} [AC4Abu] [Aeea] [Aeea] Yes q R P [NMeArg][Cha] S H K G [Oic] [Nle] [Aib] [D-4ClF] {COOH} 588 {Palm} [AC4Abu][Aeea] [Aeea] Yes q R P [NMeArg] [Cha] S H K G [Pip] [Nle] [Nip][4-Cl-F] {COOH} 597 {Palm} [AC4Abu] [Aeea] [Aeea] Yes q R P [NMeArg][Cha] S H K G [Pip] [Nle] [Aic] {COOH} 598 {Palm} [AC4Abu] [Aeea] Yes[Aeea] q R P [NMeArg] [Cha] S H K G [Pip] [Nle] P [Tic] {COOH} 599{Palm} [AC4Abu] Yes [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G [Pip][Nle] P [D-Tic] {COOH} 495 {PDA} [AC4Abu] Yes [Aeea] [Aeea] Q R P[NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 289 {Sebacicacid} [AC4Abu]Yes [Aeea] [Aeea] [hArg] r Q [hArg] P r [NMeLeu] S H K G [Oic] [pI-Phe]P [D- Bip] {COOH} 594 {St} [AC4Abu] [Aeea] [Aeea] Yes Q R P [NMeArg] L SH K G P [Nle] P [4-Cl-F] {COOH} 490 {TDA} [AC4Abu] [Aeea] Yes [Aeea][hArg] Q [hArg] P [hArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 297{TDA} [AC4Abu] [Aeea] Yes [Aeea] O F [hArg] [hArg] Q [NMeArg] P [NMeArg][Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 488 {TDA} [AC4Abu] [Aeea][Aeea] Yes q [hArg] P [hArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH}362 {TDA} [AC4Abu] [Aeea] [Aeea] Q [hArg] P Yes [NMeArg] [Cha] S H K G[Oic] [Nle] P [4-Cl-F] {COOH} 483 {TDA} [AC4Abu] [Aeea] [Aeea] Q [hArg]P Yes [NMehArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 363 {TDA} [AC4Abu][Aeea] [Aeea] Yes q [hArg] P r [Cha] S H K G [Oic] [Nle] P [4-Cl-F]{COOH} 482 {TDA} [AC4Abu] [Aeea] [Aeea] Yes Q R [aMePro] [hArg] [aMeLeu]S H K G P [Nle] P [4-Cl-F] {COOH} 502 {TDA} [AC4Abu] [Aeea] [Aeea] q R[aMePro] Yes [hArg] [BLeu] S H K G [Oic] [Nle] [aMePro] [4-Cl-F] {COOH}522 {TDA} [AC4Abu] [Aeea] [Aeea] q R [BhPro] Yes [hArg] [BLeu] S H K G[Oic] [Nle] [Aib] [4-Cl-F] {COOH} 516 {TDA} [AC4Abu] [Aeea] [Aeea] q R[BhPro] Yes [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl-F] {COOH} 517{TDA} [AC4Abu] [Aeea] [Aeea] q R [Pip] Yes [NMeArg] [Cha] S H K G [Oic][Nle] [Aib] [4-Cl-F] {COOH} 554 {TDA} [AC4Abu] [Aeea] Yes [Aeea] q R P[NMeArg] [aMeLeu] S H K G [Oic] [Nle] [BhPro] [4-Cl-F] {COOH} 500 {TDA}[AC4Abu] [Aeea] Yes [Aeea] q R P [NMeArg] [Cha] [BhSer] H K G [Oic][Nle] [Aib] [4-Cl-F] {COOH} 418 {TDA} [AC4Abu] [Aeea] [Aeea] Yes Q R P[NMeArg] [Cha] S H K G [aMePro] [Nle] P [4-Cl-F] {COOH} 551 {TDA}[AC4Abu] [Aeea] [Aeea] No q R P [NMeArg] [Cha] S H K G [aMePro] [Nle][4-Cl-F] {COOH} 382 {TDA} [AC4Abu] [Aeea] [Aeea] Yes Q R P [NMeArg][Cha] S H K G [Oic] [Nle] [Aib] [4-Cl-F] {COOH} 412 {TDA} [AC4Abu][Aeea] [Aeea] Yes Q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [aMePro][4-Cl-F] {COOH} 352 {TDA} [AC4Abu] [Aeea] [Aeea] Yes Q R P [NMeArg] L SH K G [Oic] [Nle] P [4-Cl-F] {COOH} 287 {TDA} [AC4Abu] [Aeea] Yes Yes[Aeea] Q R P [NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 526 {TDA}[AC4Abu] [Aeea] [Aeea] No q R P E [Cha] S H K G [Oic] [Nle] [Aib][4-Cl-F] {COOH} 477 {TDA} [AC4Abu] [Aeea] [Aeea] No Q R P E L S H K G P[Nle] P [4-Cl-F] {COOH}

Mast cell degranulation is proposed to occur in part by anon-immunological mechanism (Watt, A. P. Immunopharmacology, 2002, 9,421-434) related to the physical properties of peptide degranulators.For the modified apelin polypeptides, mast cell degranulation could bereduced by lowering a compound's lipophilicity and formal charge. Forinstance, acidic residues could be substituted at amino acid positionscorresponding to positions 63, 64, 66 and 68 in the apelin preprotein(SEQ ID NO: 2) as in modified polypeptides set forth in SEQ ID NOs:75-77, 477, and 526. Aliphatic residues (e.g. Nle) at the amino acidcorresponding to position 75 in SEQ ID NO: 2 and small aromatic residues(e.g. 4-Cl-F) at the amino acid corresponding to position 77 in SEQ IDNO: 2 were preferred. Pegylation or Fc protein conjugation (see Example11) at the N-terminus of the apelin polypeptide reduced mast celldegranulation as compared to the apelin polypeptide itself. For example,apelin polypeptide of SEQ ID NO: 92 was positive for inducing histaminerelease from rat mast cells. See Table 13. However, if this same peptidewas pegylated (polypeptide of SEQ ID NO: 120) or conjugated to animmunoglobulin Fc domain (immunoglobulin 4 in Example 11), the peptideno longer induced histamine release from rat mast cells. N-terminalacetylation was fairly well tolerated. In general, lipidated peptideshad increased lipophilicity and so tended to be more potent mast celldegranulators. By further reducing the formal charge (˜<1),degranulation could be avoided for lipidated peptides as well. Shorter,less lipophilic lipids had less mast cell degranulation activity asillustrated for apelin polypeptide of SEQ ID NO: 491.

Example 9. Modified Apelin Polypeptides with Improved Stability

Evaluation of in vitro plasma, liver, and kidney stability of severalmodified apelin peptides was performed.

Apelin peptides were spiked into rat plasma, liver homogenate, andkidney homogenate, which were obtained from commercial sources, to afinal concentration of 5 μM. The samples were then incubated at 37° C.over a period of 4 hr. Up to 8 time points were sampled to facilitateestimation of in vitro half-life in the three different matrices. In atypical experiment, an aliquot of 100 μL of sample was taken from theincubation vial, followed by addition of 300 μL of MeOH containinginternal standard. The quenched sample was centrifuged at 5500 g for 15min after 15 min vortex in room temperature. The supernatant was thenanalyzed by high resolution LC-MS. Accurate mass full scan mass data wasused for estimation of parent peptide level over the time course of 4hr, and the data was subsequently used for estimation of in vitrohalf-life in plasma, liver, and kidney. In vitro plasma, rat, and kidneystability results are shown below in Table 14.

TABLE 14 Stability of Modified Apelin Polypeptides in Rat Plasma, Liver,and Kidney SEQ Stability at 4 hours ID (% Remaining) NO: Sequence PlasmaLiver Kidney 6 Pyr-apelin; {Hydrogen}[PE] <1.0 <1.0 <1.0RPRLSHKGPMPF{COOH} 212 {TDA}[AC4Abu][Aeea] 39.5 >90.0 >90.0[Aeea]RQRP[hArg][NMeLeu] SHKG[Oic][pI-Phe]P[D-Bip] {COOH} 263{HDA}[AC4Abu][Aeea][Aeea] 79.3 67.6 82.6 [hArg]rQ[hArg]Pr[NMeLeu]SHKG[Oic][pI-Phe] P[D-Bip]{COOH} 237 {HDA}[AC4Abu][Aeea] 85.2 65.8 35.3[Aeea][q][hArg]P [NMeArg][Cha]SHKG [Oic][Nle]P[D-4ClF]{COOH} 16Acetyl-[hArg][r]Q[hArg]P 86.1 40.7 44.5 [r][NMeLeu]SHKG[Oic][pI-Phe]P[D-Bip]{COOH} 262 {PDA}[AC4Abu][Aeea][Aeea] >90.0 66 84.4[hArg]rQ[hArg]Pr[NMeLeu] SHKG[Oic][pI-Phe]P [D-Bip]{COOH} 261{TDA}[AC4Abu][Aeea][Aeea] >90.0 No 85.8 [hArg]rQ[hArg]Pr[NMeLeu] ResultSHKG[Oic][pI-Phe]P Obtained [D-Bip]{COOH} 76 {Acetyl-NH} [hArg] EQ >90.0 26.7 34 [hArg] P r [NMeLeu] S H K G [Oic] [pI-Phe] P [D-Bip]{COOH} 60 {Acetyl-NH} [hArg] 21.8 <1.0 0.68 Q [hArg] P [NMeArg] [Cha] SH K G [Oic] [Nle] P [4-Cl-F] {COOH} 79 {Acetyl-NH} [hArg] r Q[hArg] >90.0 <1.0 1.3 P E [NMeLeu] S H K G [Oic] [pI-Phe] P [D-Bip]{COOH} 75 {Acetyl-NH} E r Q [hArg] P r >90.0 19.5 39.9 [NMeLeu] S H K G[Oic] [pI-Phe] P [D-Bip] {COOH} 71 {Acetyl-NH} Q [hArg] P 0 35.5 0.1[NMeArg] [aMeLeu] S H K G P [Nle] P [4-Cl-F] {COOH} 61 {Acetyl-NH} Q[hArg] P 27.91 <1.0 7.575 [NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F]{COOH} 69 {Acetyl-NH} Q R [aMePro] <1.0 No <1.0 [hArg] [aMeLeu] S HResult K G P [Nle] Obtained P [4-Cl-F] {COOH} 70 {Acetyl-NH} Q r P[hArg] 86.1 84.9 No [aMeLeu] S H K G P [Nle] P Result [4-Cl-F] {COOH}Obtained 92 {Acetyl-NH} Q R P [NMeArg] 48 <1.0 <1.0 [Cha] S H K G [Oic][Nle] [Aib] [4-Cl-F] {COOH} 68 {Acetyl-NH} Q R P 59.6 <1.0 <1.0 [NMeArg]L S H K G P [Nle] P [4-Cl-F] {COOH} 66 {Acetyl-NH} R Q R P 2.4 Not Not[NMeArg] tested tested [Cha] S H K G P [Nle] P [4-Cl-F] {COOH} 62{Acetyl-NH} R Q R P 6.3 <1.0 <1.0 R L S H K G P [Nle] P [D-Bip] {COOH}515 {AdpA} [AC4Abu] 2 <1.0 6 [Aeea] [Aeea] Q r P [hArg] [aMeLeu] S H K GP [Nle] P [4-Cl-F] {COOH} 491 {Butanoyl} [AC4Abu] [Aeea] <1.0 <1.0 <1.0[Aeea] Q R P [NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 113 {H2}[MerPr] (10K mPEG 49 64 17 acetamide) Q [hArg] P [NMehArg] L S H K G P[Nle] P [4-Cl-F] {COOH} 114 {H2} [MerPr] (10K mPEG 58 >90.0 >90.0acetamide) Q R [aMePro] [hArg] [aMeLeu] S H K G P [Nle] P [4-Cl-F]{COOH} 119 {H2} [MerPr] (10K mPEG 44 >90.0 89 acetamide) Q r P [hArg][aMeLeu] S H K G P [Nle] P [4-Cl-F] {COOH} 120 {H2} [MerPr] (10K mPEG 6861 <1.0 acetamide) Q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib][4-Cl-F] {COOH} 116 {H2} [MerPr] (10K mPEG 48 >90.0 88 acetamide) Q[NMehArg] P [NMeArg] [aMeLeu] S H K G P [Nle] P [4-Cl-F] {COOH} 110 {H2}[MerPr] (10K mPEG 36 61 27 acetamide) Q R P R L S H K G P M P F {COOH}13 {H2} C M P L H S 7 <1.0 <1.0 R V P F P {COOH} 14 {H2} K L R K H N<1.0 <1.0 No [Abu] L Q R R Result [Abu] M P L H Obtained S R V P F P{COOH} 15 {H2} K L R K H N C L Q R R 17 <1.0 0 C M P L H S R V P F P{COOH} 496 {HDA} [AC4Abu] [Aeea] 86 3 <1.0 [Aeea] Q R P [NMeArg] L S H KG P [Nle] P [4-Cl-F] {COOH} 561 {hexanoyl} [AC4Abu] [Aeea] <1.0 <1.0<1.0 [Aeea] Q [NMehArg] P [NMeArg] [aMeLeu] S H K G P [Nle] P [4-Cl-F]{COOH} 562 {hexanoyl} [AC4Abu] [Aeea] >90.0 4 <1.0 [Aeea] Q R P [NMeArg][Cha] S H K G [Oic] [Nle] [Aib] [4-Cl-F] {COOH} 614 {Palm} [AC4Abu][Aeea] 90 11 9 [Aeea] q [hArg] P r [Cha] S H K G [Oic] [Nle] P [D-BhPhe]{COOH} 569 {Palm} [AC4Abu] [Aeea] >90.0 34 14 [Aeea] q R P [NMeArg][Cha] S H K G [Oic] [Nle] [Aib] [D-BhPhe] {COOH} 570 {Palm} [AC4Abu][Aeea] >90.0 32 11 [Aeea] q R P [NMeArg] [Cha] S H K G [Oic] [Nle][aMePro] [D-BhPhe] {COOH} 571 {Palm} [AC4Abu] [Aeea] >90.0 35.5 16[Aeea] q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [BhPro] [D-BhPhe] {COOH}572 {Palm} [AC4Abu] [Aeea] 89 37.5 14.5 [Aeea] q R P [NMeArg] [Cha] S HK G [Oic] [Nle] [bAla] [D-BhPhe] {COOH} 573 {Palm} [AC4Abu] [Aeea] 79 5927 [Aeea] q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [D- BhPhe] {COOH} 584{Palm} [AC4Abu] [Aeea] 62 77 6 [Aeea] q R P [NMeArg] [Cha] S H K G [Oic][Nle] [1-Ach] [4-Cl-F] {COOH} 585 {Palm} [AC4Abu] [Aeea] [Aeea] 70 65 5q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Acp] [4-Cl-F] {COOH} 587{Palm} [AC4Abu] [Aeea] [Aeea] >90.0 64 34 q R P [NMeArg] [Cha] S H K G[Oic] [Nle] [Inp] [4-Cl-F] {COOH} 588 {Palm} [AC4Abu] [Aeea][Aeea] >90.0 No No q R P [NMeArg] [Cha] Result Result S H K G [Pip][Nle] [Nip] Obtained Obtained [4-Cl-F] {COOH} 590 {Palm} [AC4Abu] [Aeea][Aeea] 86 11 4 q R P [NMeArg] [Cha] S H K G [Pip] [Nle] P [Aic] {COOH}495 {PDA} [AC4Abu] [Aeea] [Aeea] 75 <1.0 <1.0 Q R P [NMeArg] L S H K G P[Nle] P [4-Cl-F] {COOH} 508 {Subericacid} [AC4Abu] [Aeea] <1.0 <1.0 <1.0[Aeea] Q R P [NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 534 {TDA}[AC4Abu] [AC4Abu] >90.0 7 2 [Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G[Oic] [Nle] [Aib] [4-Cl-F] {COOH} 535 {TDA} [AC4Abu] [AC4Abu] 89 3 <1.0[Aeea] q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl-F] {COOH}375 {TDA} [AC4Abu] [Aeea] [Aeea] 66 12 3 [aMeLeu] R P [NMeArg] [aMeLeu]S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 552 {TDA} [AC4Abu] [Aeea][Aeea] >90.0 40 18 [BLeu] R [aMePro] [hArg] [aMeLeu] S H K G [Oic] [Nle][aMePro] [4-Cl-F] {COOH} 395 {TDA} [AC4Abu] [Aeea] [Aeea] 83 2 1 [BLeu]R P [hArg] [BLeu] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 374 {TDA}[AC4Abu] [Aeea] [Aeea] 66 11 89 [BLeu] R P [NMeArg] [aMeLeu] S H K G[Oic] [Nle] P [4-Cl-F] {COOH} 388 {TDA} [AC4Abu] [Aeea] [Aeea] 72 18 16[BLeu] R P [NMeArg] [aMeLeu] S H K G [Oic] [Nle] [aMePro] [4-Cl-F]{COOH} 394 {TDA} [AC4Abu] [Aeea] [Aeea] 86 8 2 [BLeu] R P [NMeArg][BLeu] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 393 {TDA} [AC4Abu] [Aeea][Aeea] >90.0 12 2 [BLeu] R P [NMeArg] [Cha] S H K G [Oic] [Nle] P[4-Cl-F] {COOH} 309 {TDA} [AC4Abu] [Aeea] [Aeea] >90.0 25.7 1.9 [hArg][hArg] Q [hArg] P [NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH}315 {TDA} [AC4Abu] [Aeea] [Aeea] >90.0 86.1 54.8 [hArg] [Nle] Q [hArg] Pr [NMeLeu] S H K G [Oic] [pI-Phe] P [D-Bip] {COOH} 490 {TDA} [AC4Abu][Aeea] [Aeea] 88 9 <1.0 [hArg] Q [hArg] P [hArg] [Cha] S H K G [Oic][Nle] P [4-Cl-F] {COOH} 310 {TDA} [AC4Abu] [Aeea] [Aeea] >90.0 15 1[hArg] Q [hArg] P [NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH}328 {TDA} [AC4Abu] [Aeea] [Aeea] 76 0 55 [hArg] r Q [Nle] P r [NMeLeu] SA K G [Oic] [pI-Phe] P [D-Bip] {COOH} 347 {TDA} [AC4Abu] [Aeea] [Aeea]75 3 1 [Nle] Q R P [hArg] [Cha] A A K G [Oic] [Nle] P [4-Cl-F] {COOH}342 {TDA} [AC4Abu] [Aeea] [Aeea] 76 6 2 [Nle] Q R P [hArg] [Cha] S A K G[Oic] [Nle] P [4-Cl-F] {COOH} 338 {TDA} [AC4Abu] [Aeea] [Aeea] 76 12 1[Nle] Q R P [hArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 320 {TDA}[AC4Abu] [Aeea] [Aeea] >90.0 86.5 73.4 [Nle] r Q [hArg] P r [NMeLeu] A HK G [Oic] [pI-Phe] P [D-Bip] {COOH} 314 {TDA} [AC4Abu] [Aeea][Aeea] >90.0 77.5 71 [Nle] r Q [hArg] P r [NMeLeu] S H K G [Oic][pI-Phe] P [D-Bip] {COOH} 307 {TDA} [AC4Abu] [Aeea] [Aeea] >90.0 66.813.8 O F [hArg] [hArg] [BLeu] [hArg] P [hArg] [Cha] H K G [Oic] S[Nle] P[4-Cl-F] {COOH} 297 {TDA} [AC4Abu] [Aeea] [Aeea] >90.0 40.3 <1.0 O F[hArg] [hArg] Q [NMeArg] P [NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F]{COOH} 550 {TDA} [AC4Abu] [Aeea] [Aeea] >90.0 2 <1.0 Q [BhPro] [NMeArg][Cha] S H K G [Oic] [Nle] [Aib] [4-Cl-F] {COOH} 488 {TDA} [AC4Abu][Aeea] [Aeea] 65 3 <1.0 q [hArg] P [hArg] [Cha] S H K G [Oic] [Nle] P[4-Cl-F] {COOH} 358 {TDA} [AC4Abu] [Aeea] [Aeea] 85 2.5 0.5 Q [hArg] P[hArg] L S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 357 {TDA} [AC4Abu] [Aeea][Aeea] 56 0 0 Q [hArg] P [hArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 359{TDA} [AC4Abu] [Aeea] [Aeea] 62 3 2 Q [hArg] P [NMeArg] [aMeLeu] S H K G[Oic] [Nle] P [4-Cl-F] {COOH} 473 {TDA} [AC4Abu] [Aeea] [Aeea] 82 <1.0<1.0 Q [hArg] P [NMeArg] [aMeLeu] S H K G P [Nle] P [2Nal] {COOH} 383{TDA} [AC4Abu] [Aeea] [Aeea] 31 <1.0 <1.0 Q [hArg] P [NMeArg] [BLeu] S HK G P [Nle] P [4-Cl-F] {COOH} 362 {TDA} [AC4Abu] [Aeea] [Aeea] 79 39 2.5Q [hArg] P [NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 353{TDA} [AC4Abu] [Aeea] [Aeea] 20 0 0 Q [hArg] P [NMeArg] L S H K G P[Nle] P [4-Cl-F] {COOH} 483 {TDA} [AC4Abu] [Aeea] [Aeea] 37 <1.0 <1.0 Q[hArg] P [NMehArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 372 {TDA}[AC4Abu] [Aeea] [Aeea] 71 4 2 q [hArg] P r [Cha] S A K G [Oic] [Nle] P[4-Cl-F] {COOH} 371 {TDA} [AC4Abu] [Aeea] [Aeea] 90 81 28 q [hArg] P r[Cha] S H K [Tle] [Oic] [Nle] P [4-Cl-F] {COOH} 363 {TDA} [AC4Abu][Aeea] [Aeea] 78 No 1 q [hArg] P r [Cha] S H K G Result [Oic] [Nle] P[4-Cl-F] {COOH} Obtained 373 {TDA} [AC4Abu] [Aeea] [Aeea] No No 64 q[hArg] P r [Cha] S Y K G Result Result [Oic] [Nle] P [4-Cl-F] {COOH}Obtained Obtained 397 {TDA} [AC4Abu] [Aeea] [Aeea] 87 2 1 Q [NMeArg][aMeLeu] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 484 {TDA} [AC4Abu] [Aeea][Aeea] 79 <1.0 <1.0 Q [NMehArg] P [NMeArg] [aMeLeu] S H K G P [Nle] P[4-Cl-F] {COOH} 475 {TDA} [AC4Abu] [Aeea] [Aeea] 33 <1.0 <1.0 Q E P[NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 482 {TDA} [AC4Abu] [Aeea][Aeea] 62 <1.0 <1.0 Q R [aMePro] [hArg] [aMeLeu] S H K G P [Nle] P[4-Cl-F] {COOH} 392 {TDA} [AC4Abu] [Aeea] [Aeea] 73 82 <1.0 Q R [aMePro][hArg] [BLeu] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 501 {TDA} [AC4Abu][Aeea] [Aeea] >90.0 <1.0 <1.0 q R [aMePro] [hArg] [BLeu] S H K G [Pip][Nle] [4-Cl-F] {COOH} 502 {TDA} [AC4Abu] [Aeea] [Aeea] >90.0 34 19 q R[aMePro] [hArg] [BLeu] S H K G [Oic] [Nle] [aMePro] [4-Cl-F] {COOH} 503{TDA} [AC4Abu] [Aeea] [Aeea] 85 7 2 q R [aMePro] [hArg] [BLeu] S H K G[Pip] [Nle] [aMePro] [4-Cl-F] {COOH} 504 {TDA} [AC4Abu] [Aeea][Aeea] >90.0 11 4 q R [aMePro] [hArg] [BLeu] S H K G [aMePro] [Nle][aMePro] [4-Cl-F] {COOH} 505 {TDA} [AC4Abu] [Aeea] [Aeea] 24 No No q R[aMePro] [hArg] [BLeu] S H Result Result K G [Oic] [Nle] ObtainedObtained [Aib] [4-Cl-F] {COOH} 522 {TDA} [AC4Abu] [Aeea] >90.0 15 7[Aeea] q R [BhPro] [hArg] [BLeu] S H K G [Oic] [Nle] [Aib] [4-Cl-F]{COOH} 553 {TDA} [AC4Abu] [Aeea] [Aeea] >90.0 29 15 q R [BhPro] [NMeArg][aMeLeu] S H K G [Oic] [Nle] [aMePro] [4-Cl-F] {COOH} 516 {TDA} [AC4Abu][Aeea] [Aeea] >90.0 6 8 q R [BhPro] [NMeArg] [Cha] S H K G [Oic] [Nle][Aib] [4-Cl-F] {COOH} 396 {TDA} [AC4Abu] [Aeea] [Aeea] >90.0 <1.0 <1.0 QR [NMeArg] [aMeLeu] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 517 {TDA}[AC4Abu] [Aeea] [Aeea] >90.0 19 <1.0 q R [Pip] [NMeArg] [Cha] S H K G[Oic] [Nle] [Aib] [4-Cl-F] {COOH} 476 {TDA} [AC4Abu] [Aeea] [Aeea] 40<1.0 <1.0 Q R E [NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 360 {TDA}[AC4Abu] [Aeea] [Aeea] 81.5 2.5 2 Q R P [hArg] [aMeLeu] S H K G [Oic][Nie] P [4-Cl-F] {COOH} 485 {TDA} [AC4Abu] [Aeea] [Aeea] 74 <1.0 <1.0 Qr P [hArg] [aMeLeu] S H K G P [Nle] P [4-Cl-F] {COOH} 378 {TDA} [AC4Abu][Aeea] [Aeea] 58 2 75 q R P [hArg] [BLeu] S H K G [Oic] [Nle] P [4-Cl-F]{COOH} 308 {TDA} [AC4Abu] [Aeea] [Aeea] 76 6.2 <1.0 Q R P [hArg] [Cha] SH K G [Oic] [Nle] P [4-Cl-F] {COOH} 356 {TDA} [AC4Abu] [Aeea] [Aeea] 872 0 Q R P [hArg] L S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 355 {TDA}[AC4Abu] [Aeea] [Aeea] 64 0 0 Q R P [hArg] L S H K G P [Nle] P [4-Cl-F]{COOH} 387 {TDA} [AC4Abu] [Aeea] [Aeea] 73 4 1 Q R P [NMeArg] [aMeLeu] SH K G [aMePro] [Nle] P [4-Cl-F] {COOH} 376 {TDA} [AC4Abu] [Aeea] [Aeea]83 10 13 Q R P [NMeArg] [aMeLeu] S H K G [Oic] [Nle] [aMePro] [4-Cl-F]{COOH} 385 {TDA} [AC4Abu] [Aeea] [Aeea] 65 5 4 Q R P [NMeArg] [aMeLeu] SH K G [Oic] [Nle] P [4-Cl-F] {COOH} 386 {TDA} [AC4Abu] [Aeea] [Aeea] 586 4 q R P [NMeArg] [aMeLeu] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 389{TDA} [AC4Abu] [Aeea] [Aeea] <1.0 <1.0 4 Q R P [NMeArg] [aMeLeu] S H K G[Oic] [Nle] [4-Cl-F] {COOH} 554 {TDA} [AC4Abu] [Aeea] [Aeea] >90.0 43 25q R P [NMeArg] [aMeLeu] S H K G [Oic] [Nle] [BhPro] [4-Cl-F] {COOH} 555{TDA} [AC4Abu] [Aeea] [Aeea] >90.0 <1.0 <1.0 q R P [NMeArg] [aMeLeu] S HK G [Pip] [Nle] [aMePro] [4-Cl-F] {COOH} 457 {TDA} [AC4Abu] [Aeea][Aeea] 62 <1.0 <1.0 Q R P [NMeArg] [aMeLeu] S H K G P [Nle] P [4-Cl-F]{COOH} 422 {TDA} [AC4Abu] [Aeea] [Aeea] 37 <1.0 <1.0 Q R P [NMeArg][BhLeu] S H K G P [Nle] P [4-Cl-F] {COOH} 391 {TDA} [AC4Abu] [Aeea][Aeea] 2 5 2 Q R P [NMeArg] [BLeu] S H K G [Oic] [Nle] P [4-Cl-F] {COOH}500 {TDA} [AC4Abu] [Aeea] [Aeea] >90.0 13 <1.0 q R P [NMeArg] [Cha][BhSer] H K G [Oic] [Nle] [Aib] [4-Cl-F] {COOH} 530 {TDA} [AC4Abu][Aeea] [Aeea] >90.0 3 <1.0 q R P [NMeArg] [Cha] S A K G [Oic] [Nle][Aib] [4-Cl-F] {COOH} 418 {TDA} [AC4Abu] [Aeea] [Aeea] >90.0 <1.0 8 Q RP [NMeArg] [Cha] S H K G [aMePro] [Nle] P [4-Cl-F] {COOH} 498 {TDA}[AC4Abu] [Aeea] [Aeea] >90.0 >90.0 23 q R P [NMeArg] [Cha] S H K G[aMePro] [Nle] [Aib] [4-Cl-F] {COOH} 377 {TDA} [AC4Abu] [Aeea] [Aeea] 654 99 q R P [NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 379{TDA} [AC4Abu] [Aeea] [Aeea] 92 2 1 Q R P [NMeArg] [Cha] S H K G [Oic][Nle] P [BhPhe] {COOH} 380 {TDA} [AC4Abu] [Aeea] [Aeea] 97 2 1 Q R P[NMeArg] [Cha] S H K G [Oic] [Nle] P [D-BhPhe] {COOH} 382 {TDA} [AC4Abu][Aeea] [Aeea] 91 6 2 Q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib][4-Cl-F] {COOH} 390 {TDA} [AC4Abu] [Aeea] [Aeea] 60 4 4 Q R P [NMeArg][Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 412 {TDA} [AC4Abu] [Aeea][Aeea] 77 76 3 Q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [aMePro][4-Cl-F] {COOH} 416 {TDA} [AC4Abu] [Aeea] [Aeea] 57.667 No No Q R P[NMeArg] [Cha] S H K Result Result G [Pip] [Nle] P Obtained Obtained[4-Cl-F] {COOH} 499 {TDA} [AC4Abu] [Aeea] [Aeea] >90.0 <1.0 <1.0 q R P[NMeArg] [Cha] S H K G [Pip] [Nle] [Aib] [4-Cl-F] {COOH} 361 {TDA}[AC4Abu] [Aeea] [Aeea] 34.5 1.5 0.5 Q R P [NMeArg] [Cha] S H K G P [Nle]P [4-Cl-F] {COOH} 437 {TDA} [AC4Abu] [Aeea] [Aeea] 62 6 5 Q R P [NMeArg][Cha] S V K G [Oic] [Nle] P [4-Cl-F] {COOH} 531 {TDA} [AC4Abu] [Aeea][Aeea] >90.0 13 <1.0 q R P [NMeArg] [Cha] S Y K G [Oic] [Nle] [Aib][4-Cl-F] {COOH} 464 {TDA} [AC4Abu] [Aeea] [Aeea] 21 <1.0 <1.0 Q R P[NMeArg] L [bAla] H K G P [Nle] P [4-Cl-F] {COOH} 467 {TDA} [AC4Abu][Aeea] [Aeea] 27 87 83 Q R P [NMeArg] L [NhSerG] H K G P [Nle] P[4-Cl-F] {COOH} 469 {TDA} [AC4Abu] [Aeea] [Aeea] 36 <1.0 <1.0 Q R P[NMeArg] L S [rHis] K G P [Nle] P [4-Cl-F] {COOH} 424 {TDA} [AC4Abu][Aeea] [Aeea] 2 <1.0 <1.0 Q R P [NMeArg] L S H [bAla] G P [Nle] P[4-Cl-F] {COOH} 352 {TDA} [AC4Abu] [Aeea] [Aeea] 88 0 0 Q R P [NMeArg] LS H K G [Oic] [Nle] P [4-Cl-F] {COOH} 402 {TDA} [AC4Abu] [Aeea] [Aeea]87 <1.0 <1.0 Q R P [NMeArg] L S H K G P [Nle] [4-Cl-F] {COOH} 401 {TDA}[AC4Abu] [Aeea] [Aeea] 6 1 3 Q R P [NMeArg] L S H K G P [Nle] [aMePro][4-Cl-F] {COOH} 425 {TDA} [AC4Abu] [Aeea] [Aeea] >90.0 <1.0 <1.0 Q R P[NMeArg] L S H K G P [Nle] [bAla] [4-Cl-F] {COOH} 426 {TDA} [AC4Abu][Aeea] [Aeea] 33 <1.0 <1.0 Q R P [NMeArg] L S H K G P [Nle] [BhPro][4-Cl-F] {COOH} 287 {TDA} [AC4Abu] [Aeea] [Aeea] 18 <1.0 <1.0 Q R P[NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 384 {TDA} [AC4Abu] [Aeea][Aeea] 25 <1.0 <1.0 Q r P [NMehArg] [BLeu] S H K G P [Nle] P [4-Cl-F]{COOH} 471 {TDA} [AC4Abu] [Aeea] [Aeea] 52 <1.0 <1.0 Q R P [NMehArg] L SH K G P [Nle] P [2Nal] {COOH} 526 {TDA} [AC4Abu] [Aeea] [Aeea] >90.0 5<1.0 q R P E [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl-F] {COOH} 477 {TDA}[AC4Abu] [Aeea] [Aeea] 64 <1.0 2 Q R P E L S H K G P [Nle] P [4-Cl-F]{COOH} 348 {TDA} [AC4Abu] [Aeea] 75 5 1 [Aeea] R Q [Nle] P [hArg] [Cha]A A K G [Oic] [Nle] P [4-Cl-F] {COOH} 343 {TDA} [AC4Abu] [Aeea] 80 6 2[Aeea] R Q [Nle] P [hArg] [Cha] S A K G [Oic] [Nle] P [4-Cl-F] {COOH}564 {TDA} [AC4Abu] [Aeea] >90.0 2 <1.0 [Aeea] R Q [Nle] P [hArg] [Cha] SA K G [Pip] [Nle] P [4-Cl-F] {COOH} 339 {TDA} [AC4Abu] 93 13 2 [Aeea][Aeea] R Q [Nle] P [hArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH}344 {TDA} [AC4Abu] [Aeea] 88 6 0 [Aeea] RQRP [Nle] [Cha] A H K G [Oic][Nle] P [4-Cl-F] {COOH} 539 {TDA} [AC4Abu] [NPeg11] 63 3 <1.0 q R P[NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl-F] {COOH} 538 {TDA} d[Aeea] [Aeea] q R P 73 2 <1.0 [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib][4-Cl-F] {COOH} 537 {TDA} E [Aeea] [Aeea] q R P >90.0 15 <1.0 [NMeArg][Cha] S H K G [Oic] [Nle] [Aib] [4-Cl-F] {COOH}

Based on the results of these stability studies, sites of metabolismwere identified within the modified apelin polypeptides and these siteswere substituted with metabolically more stable amino acids. Non-naturalamino acids were introduced through chemical synthesis in order to blockmetabolism or reduce recognition by peptidases. An N-Methyl amino acidat positions corresponding to amino acids 66 (NMeArg66), 68 (NMeArg68),and 69 (NMeLeu69) in SEQ ID NO: 2, an N-alkyl amino acid at positioncorresponding to amino acid 70 (NhSerG70) in SEQ ID NO: 2, and analpha-methyl amino acid at positions corresponding to amino acids 67(aMePro67), 69 (aMeLeu69), 74 (aMePro74), and 76 (aMePro76) in SEQ ID N:2 can sterically hinder access of peptidases to bonds in the apelinpeptides. D-amino acids at positions corresponding to amino acids 64,65, 66, and 68 in SEQ ID NO: 2 (r64, q65, r66, r68) and beta-amino acidsat positions corresponding to amino acids 65, 69, 70, 76, and 77 in SEQID NO: 2 (BLeu65, BLeu69, BhSer70, BhPro76, D-BhPhe77) can avoidrecognition of peptidases that cleave endogenous peptides containingL-amino acids. ψ(CH₂NH)-reduced amide bond amino acids, for example at aposition corresponding to amino acid 75 in SEQ ID NO: 2 (rNle75),replace an amide bond with a more stable amine bond. Cyclic peptides arenot recognized by exopeptidases and sterically hinder access by otherpeptidases. Oxidative metabolism at methionine75 in the native sequence(amino acid positions relative to SEQ ID NO: 2) was avoided bysubstitution with norleucine75. The combination of NMeLeu, pl-Phe, andD-Bip at positions corresponding to amino acids 69, 75, and 77,respectively, in SEQ ID NO: 2 gave particularly stable peptides.Conjugation with lipid or PEG improved in vitro stability of thepeptides and reduced clearance in vivo.

An in vivo pharmacokinetics analysis in rat was conducted for fourlipidated apelin peptides (SEQ ID NOs: 286, 376, 379, and 382) accordingto the methods described in Example 6. Table 15 below summarizes theresults of the pharmacokinetic study.

TABLE 15 Rat PK Analysis for Modified Apelin Polypeptides Apelin S.C.Bolus Peptide Dose AUC_(inf) CL t1/2 SEQ ID NO: (mg/kg) (μM · h) (L/(h ·kg)) (h) 286 0.1 0.378 0.099 2.31 0.5 1.059 0.200 2.59 376 0.1 0.2720.164 0.446 0.5 0.090 2.50 1.48 379 0.1 0.145 0.304 0.261 0.5 0.052 4.282.27 382 0.1 0.336 0.135 0.485 0.5 0.0961 2.40 4.10

Example 10. Modified Apelin Polypeptides with Improved Potency

In some cases, modification of the apelin polypeptides to increasestability led to a decrease in potency of the peptides for activatingthe APJ receptor. To improve the APJ agonist potency of the stabilizedpeptides, a further SAR study was conducted. The modified apelinpolypeptides in Table 16 below were tested for APJ agonist activityusing the GTPγS assay as described in Example 3. EC50 values areprovided for each modified polypeptide for activation of the human andrat APJ receptors.

The results show that potency and efficacy of the peptides could beobtained by preserving features of the sidechains of the nativeapelin-13 sequence (SEQ ID NO: 4), while the peptide backbone wasmodified for stability. At the N-terminus of the modified polypeptide,the sequence: hArg hArg Q hArg P (SEQ ID NO: 715) at positionscorresponding to amino acids 63 to 67 of SEQ ID NO: 2 provided goodpotency. At positions corresponding to amino acids 68 and 69 of SEQ IDNO: 2, the following pairs of substitutions gave potent agonists: (i)NMeArg and aMeLeu, (ii) hArg and BLeu, (iii) hArg and aMeLeu, and (iv)NMehArg and L. Positions within the modified polypeptides correspondingto amino acids 70 to 73 of SEQ ID NO: 2 were less amenable tosubstitutions, however BhSer70, NhSerG70, Y71 and NLysG72 weretolerated. At the amino acid position in the modified polypeptidecorresponding to amino acid 74 of SEQ ID NO: 2, Oic and aMePro werepreferred for achieving potent peptides, and Pip at this position alsotended to improve potency. At the amino acid position in the modifiedpolypeptide corresponding to amino acid 75 of SEQ ID NO: 2, Nle or rNlegave potent compounds. C terminal residues (positions corresponding toamino acids 76 and 77 of SEQ ID NO: 2) were critical to both activityand stability. BhPro, aMePro, or Aib at the position corresponding toamino acid 76 and D-BhPhe or 4-Cl-F at the position corresponding toamino acid 77 gave a good balance of potency and stability. N-terminallipid conjugation had little effect on potency or efficacy. PEG and Fcconjugates (see Example 11) were not soluble in DMSO and in vitro assayswere run from aqueous stocks. In the absence of DMSO, potency andefficacy read-outs were generally reduced in the GTPγS assay. However,relative to [MerPr](10K-mPEGacetamideReg)-apelin13 run under the sameconditions (hAPJ EC50 0.21 μM; rAPJ EC50 0.39 μM), PEG and Fc conjugatesof the modified polypeptides were potent full APJ agonists.

TABLE 16 APJ Agonist Activity of Modified Apelin Polypeptides EC50 EC50Human Rat SEQ APJ APJ ID Receptor Receptor NO: SEQUENCE (μM) (μM) 88{Acetyl-NH} [AC4Abu] [Aeea] 0.0002195 0.000388 [Aeea] Q R P [NMeArg] L SH K G P [Nle] P [4-Cl-F] {COOH} 76 {Acetyl-NH} [hArg] E Q [hArg]0.001845 0.000994 P r [NMeLeu] S H K G [Oic] [pI-Phe] P [D-Bip] {COOH}60 {Acetyl-NH} [hArg] Q [hArg] P 0.00022412 0.0044764 [NMeArg] [Cha] S HK G [Oic] [Nle] P [4-Cl-F] {COOH} 78 {Acetyl-NH} [hArg] r Q [hArg]0.000882 0.000871 E r [NMeLeu] S H K G [Oic] [pI-Phe] P [D-Bip] {COOH}79 {Acetyl-NH} [hArg] r Q [hArg] 0.000678 0.000341 P E [NMeLeu] S H K G[Oic] [pI-Phe] P [D-Bip] {COOH} 81 {Acetyl-NH} [hArg] r Q [hArg] 0.083850.0706 P r [NMeLeu] E H K G [Oic] [pI-Phe] P [D-Bip] {COOH} 82{Acetyl-NH} [hArg] r Q [hArg] 0.02315 0.0539 P r [NMeLeu] S E K G [Oic][pI-Phe] P [D-Bip] {COOH} 83 {Acetyl-NH} [hArg] r Q [hArg] 0.0545 0.0847P r [NMeLeu] S H E G [Oic] [pI-Phe] P [D-Bip] {COOH} 84 {Acetyl-NH}[hArg] r Q [hArg] 0.01815 0.0893 P r [NMeLeu] S H K E [Oic] [pI-Phe] P[D-Bip] {COOH} 87 {Acetyl-NH} [hArg] r Q [hArg] 0.05265 0.0589 P r[NMeLeu] S H K G [Oic] [pI-Phe] E [D-Bip] {COOH} 86 {Acetyl-NH} [hArg] rQ [hArg] >4.17 >4.17 P r [NMeLeu] S H K G [Oic] E P [D-Bip] {COOH} 85{Acetyl-NH} [hArg] r Q [hArg] >4.17 >4.17 P r [NMeLeu] S H K G E[pI-Phe] P [D-Bip] {COOH} 80 {Acetyl-NH} [hArg] r Q [hArg] 0.12730.06865 P r E S H K G [Oic] [pI-Phe] P [D-Bip] {COOH} 77 {Acetyl-NH}[hArg] r Q E P r 0.00492 0.002845 [NMeLeu] S H K G [Oic] [pI-Phe] P[D-Bip] {COOH} 75 {Acetyl-NH} E r Q [hArg] 0.00156 0.0010805 P r[NMeLeu] S H K G [Oic] [pI-Phe] P [D-Bip] {COOH} 74 {Acetyl-NH} Q [hArg]0.00048 0.0173 [NMehArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 71{Acetyl-NH} Q [hArg] P 0.0000921 0.00275 [NMeArg] [aMeLeu] S H K G P[Nle] P [4-Cl-F] {COOH} 61 {Acetyl-NH} Q [hArg] P 0.00015933 0.000189[NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 69 {Acetyl-NH} Q R[aMePro] 0.0000417 0.0003135 [hArg] [aMeLeu] S H K G P [Nle] P [4-Cl-F]{COOH} 72 {Acetyl-NH} Q R [NMeArg] 0.0013575 0.04065 L S H K G P [Nle] P[4-Cl-F] {COOH} 73 {Acetyl-NH} Q R [NMehArg] 0.0018115 0.047225 L S H KG P [Nle] P [4-Cl-F] {COOH} 70 {Acetyl-NH} Q r P [hArg] 0.00019250.009735 [aMeLeu] S H K G P [Nle] P [4-Cl-F] {COOH} 93 {Acetyl-NH} q R P[NMeArg] 0.0002295 0.0007905 [Cha] S H K G [aMePro] [Nle] [Aib] [4-Cl-F]{COOH} 92 {Acetyl-NH} Q R P [NMeArg] 0.000679 0.0032275 [Cha] S H K G[Oic] [Nle] [Aib] [4-Cl-F] {COOH} 68 {Acetyl-NH} Q R P [NMeArg]0.0000842 0.00038429 L S H K G P [Nle] P [4-Cl-F] {COOH} 91 {Acetyl-NH}R L I E D I C L P R 0.0000357 0.000124 W G C L W [Aeea] [Aeea] Q R P[NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 90 {Acetyl-NH} R L I E D IC L P R 0.0001395 0.0006325 W G C L W E D D [Aeea] [Aeea] Q R P [NMeArg]L S H K G P [Nle] P [4-Cl-F] {COOH} 66 {Acetyl-NH} R Q R P [NMeArg]0.000040467 0.00015567 [Cha] S H K G P [Nle] P [4-Cl-F] {COOH} 67{Acetyl-NH} R Q R P [NMeArg] 0.00018467 0.0014023 [NMeLeu] S H K G P[Nle] P [4-Cl-F] {COOH} 65 {Acetyl-NH} R Q R P [NMeArg] 0.00003020.00012227 L S H K G P [Nle] P [4-Cl-F] {COOH} 63 {Acetyl-NH} R Q R P R[Cha] 0.000035967 0.00016433 S H K G P [Nle] P [4-Cl-F] {COOH} 64{Acetyl-NH} R Q R P R 0.000055533 0.00041067 [NMeLeu] S H K G P [Nle] P[4-Cl-F] {COOH} 62 {Acetyl-NH} R Q R P R L S H K 0.00030333 0.0034333 GP [Nle] P [D-Bip] {COOH} 94 {Acetyl-NH} S D F Y K R L I N 0.00134150.00133 K A K [Aeea] [Aeea] [hArg] r Q [hArg] P r [NMeLeu] S H K G [Oic][pI-Phe] P [D-Bip] {COOH} 89 {Acetyl-NH} S D F Y K R L I N 0.00003890.00010095 K A K [Aeea] [Aeea] Q R P [NMeArg] L S H K G P [Nle] P[4-Cl-F] {COOH} 515 {AdpA} [AC4Abu] [Aeea] [Aeea] 0.001815 0.005935 Q rP [hArg] [aMeLeu] S H K G P [Nle] P [4-Cl-F] {COOH} 461 {AdpA} [AC4Abu][Aeea] [Aeea] 0.0002029 0.00099975 Q R P [NMeArg] L S H K G P [Nle] P[4-Cl-F] {COOH} 491 {Butanoyl} [AC4Abu] [Aeea] 0.00006745 0.000453[Aeea] Q R P [NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 349 {DDDA}[AC4Abu] [Aeea] 0.06395 0.047833 [Aeea] Q [hArg] P r [NMeLeu] S H K G[Oic] [pI-Phe] P [D-Bip] {COOH} 510 {DDDA} [AC4Abu] [Aeea] 0.00020750.000903 [Aeea] Q R P [NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 494{Decanoyl} [AC4Abu] [Aeea] 0.00002215 0.0001465 [Aeea] Q R P [NMeArg] LS H K G P [Nle] P [4-Cl-F] {COOH} 591 {Dodecanoyl-NH} [AC4Abu] 0.00047550.00184 [Aeea] [Aeea] Q R P [NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH}111 {H2} [MerPr] (10K mPEG 1.095 0.777 acetamide) q [hArg] P [NMeArg][Cha] S H K G [Oic] [Nle] P [D-4ClF] {COOH} 112 {H2} [MerPr] (10K mPEG0.00232 0.010435 acetamide) Q [hArg] P [NMeArg] [aMeLeu] S H K G P [Nle]P [4-Cl-F] {COOH} 113 {H2} [MerPr] (10K mPEG 0.01214 0.0493 acetamide) Q[hArg] P [NMehArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 114 {H2} [MerPr](10K mPEG 0.000848 0.003455 acetamide) Q R [aMePro] [hArg] [aMeLeu] S HK G P [Nle] P [4-Cl-F] {COOH} 117 {H2} [MerPr] (10K mPEG 0.01965 0.11715acetamide) Q R P [hArg] [aMeLeu] S H K G P [Nle] P [4-Cl-F] {COOH} 119{H2} [MerPr] (10K mPEG 0.0256 0.141 acetamide) Q r P [hArg] [aMeLeu] S HK G P [Nle] P [4-Cl-F] {COOH} 120 {H2} [MerPr] (10K mPEG 0.0632 0.2585acetamide) Q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl-F]{COOH} 115 {H2} [MerPr] (10K mPEG 0.00166 0.00649 acetamide) Q R P[NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 116 {H2} [MerPr] (10K mPEG0.009715 0.0408 acetamide) Q [NMehArg] P [NMeArg] [aMeLeu] S H K G P[Nle] P [4-Cl-F] {COOH} 110 {H2} [MerPr] (10K mPEG 0.21 0.3915acetamide) Q R P R L S H K G P M P F {COOH} 13 {H2} C M P L H S R V P FP 0.0703 0.50367 {COOH} 548 {H2} H A E G T F T S D V S S Y L E G Q A A K(AC4Abu-Palm) E F I A W L V R G R G {COOH} 12 {H2} K F R R Q R P R L S H0.0014167 0.0058083 K G P M P {COOH} 14 {H2} K L R K H N [Abu] L Q0.000301 0.032433 R R [Abu] M P L H S R V P F P {COOH} 15 {H2} K L R K HN C L Q R R 0.00013123 0.016087 C M P L H S R V P F P {COOH} 576 {HDA}[AC4Abu] [Aeea] [Aeea] 0.003335 0.005165 q R P [NMeArg] [Cha] S H K G[Oic] [Nle] P [D-4ClF] {COOH} 496 {HDA} [AC4Abu] [Aeea] [Aeea] 0.00008740.0004815 Q R P [NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 506{hexanoyl} [AC4Abu] [Aeea] 0.0034925 0.013238 [Aeea] [Aeea] [hArg] Q[hArg] P [NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 286{hexanoyl} [AC4Abu] [Aeea] 0.00162 0.003074 [Aeea] [hArg] r Q [hArg] P r[NMeLeu] S H K G [Oic] [pI-Phe] P [D-Bip] {COOH} 563 {hexanoyl} [AC4Abu][Aeea] 0.0043 0.012745 [Aeea] [hArg] r Q [hArg] P r [NMeLeu] S H K G[Pip] [pI-Phe] P [D-Bip] {COOH} 561 {hexanoyl} [AC4Abu] [Aeea] 0.00120830.0059375 [Aeea] Q [NMehArg] P [NMeArg] [aMeLeu] S H K G P [Nle] P[4-Cl-F] {COOH} 560 {hexanoyl} [AC4Abu] [Aeea] 0.0000553 0.000274 [Aeea]Q R [aMePro] [hArg] [aMeLeu] S H K G P [Nle] P [4-Cl-F] {COOH} 514{hexanoyl} [AC4Abu] [Aeea] 0.000485 0.00669 [Aeea] Q r P [hArg] [aMeLeu]S H K G P [Nle] P [4-Cl-F] {COOH} 559 {hexanoyl} [AC4Abu] [Aeea] 0.001160.009155 [Aeea] Q R P [hArg] [aMeLeu] S H K G P [Nle] P [4-Cl-F] {COOH}562 {hexanoyl} [AC4Abu] [Aeea] 0.00385 0.016275 [Aeea] Q R P [NMeArg][Cha] S H K G [Oic] [Nle] [Aib] [4-Cl-F] {COOH} 492 {hexanoyl} [AC4Abu][Aeea] 0.00003755 0.000147 [Aeea] Q R P [NMeArg] L S H K G P [Nle] P[4-Cl-F] {COOH} 566 {hexanoyl} [Ahx] [AC4Abu] 0.0011335 0.00165 [Aeea][Aeea] [hArg] r Q [hArg] P r [NMeLeu] S H K G [Oic] [pI-Phe] P [D-Bip]{COOH} 512 {HexDDA} [AC4Abu] [Aeea] 0.0006585 0.004595 [Aeea] Q R P[NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 592 {Myristyl-NH} [AC4Abu][Aeea] 0.0002765 0.0005975 [Aeea] Q R P [NMeArg] L S H K G P [Nle] P[4-Cl-F] {COOH} 493 {Oc} [AC4Abu] [Aeea] [Aeea] Q 0.00012008 0.000647 RP [NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 513 {ODDA} [AC4Abu][Aeea] 0.0008815 0.00612 [Aeea] Q R P [NMeArg] L S H K G P [Nle] P[4-Cl-F] {COOH} 290 {ODDA} [AC4Abu] [Aeea] 0.0401 0.17285 [Aeea] Q R P RL S H K G P M P F {COOH} 614 {Palm} [AC4Abu] [Aeea] [Aeea] 0.0012450.010275 q [hArg] P r [Cha] S H K G [Oic] [Nle] P [D-BhPhe] {COOH} 646{Palm} [AC4Abu] [Aeea] [Aeea] 0.0002695 0.0009935 q [NPipG] P [NMeArg][Cha] S H K G [Pip] [Nle] [Aib] [4-Cl-F] {COOH} 627 {Palm} [AC4Abu][Aeea] [Aeea] 0.0002435 0.000641 q R P [NMeArg] [aMeLeu] [aMeS] H K G[Oic] [Nle] [BhPro] [4-Cl-F] {COOH} 628 {Palm} [AC4Abu] [Aeea] [Aeea]0.0003095 0.0007735 q R P [NMeArg] [aMeLeu] [BLeu] H K G [Oic] [Nle][BhPro] [4-Cl-F] {COOH} 629 {Palm} [AC4Abu] [Aeea] [Aeea] 0.0024450.005305 q R P [NMeArg] [aMeLeu] S [Bip] K G [Oic] [Nle] [BhPro][4-Cl-F] {COOH} 630 {Palm} [AC4Abu] [Aeea] [Aeea] 0.000203 0.0004865 q RP [NMeArg] [aMeLeu] S [Dap] K G [Oic] [Nle] [BhPro] [4-Cl-F] {COOH} 636{Palm} [AC4Abu] [Aeea] [Aeea] 0.001587 0.00365 q R P [NMeArg] [aMeLeu] SH [3Pal] G [Oic] [Nle] [BhPro] [4-Cl-F] {COOH} 637 {Palm} [AC4Abu][Aeea] [Aeea] 0.00405 0.014155 q R P [NMeArg] [aMeLeu] S H [4-F-F] G[Oic] [Nle] [BhPro] [4-Cl-F] {COOH} 635 {Palm} [AC4Abu] [Aeea] [Aeea]0.0016185 0.006645 q R P [NMeArg] [aMeLeu] S H [aMeOrn] G [Oic] [Nle][BhPro] [4-Cl-F] {COOH} 638 {Palm} [AC4Abu] [Aeea] [Aeea] 0.0032 0.00932q R P [NMeArg] [aMeLeu] S H [Dab] G [Oic] [Nle] [BhPro] [4-Cl-F] {COOH}639 {Palm} [AC4Abu] [Aeea] [Aeea] 0.00328 0.0154 q R P [NMeArg] [aMeLeu]S H [Dap] G [Oic] [Nle] [BhPro] [4-Cl-F] {COOH} 640 {Palm} [AC4Abu][Aeea] [Aeea] 0.004025 0.0252 q R P [NMeArg] [aMeLeu] S H [Igl] G [Oic][Nle] [BhPro] [4-Cl-F] {COOH} 632 {Palm} [AC4Abu] [Aeea] [Aeea] 0.003020.022 q R P [NMeArg] [aMeLeu] S H [NPipG] G [Oic] [Nle] [BhPro] [4-Cl-F]{COOH} 641 {Palm} [AC4Abu] [Aeea] [Aeea] 0.00058 0.00366 q R P [NMeArg][aMeLeu] S H K [Sar] [Oic] [Nle] [BhPro] [4-Cl-F] {COOH} 631 {Palm}[AC4Abu] [Aeea] [Aeea] 0.0003385 0.0006985 q R P [NMeArg] [aMeLeu] S h KG [Oic] [Nle] [BhPro] [4-Cl-F] {COOH} 634 {Palm} [AC4Abu] [Aeea] [Aeea]0.0001645 0.002205 q R P [NMeArg] [aMeLeu] S H O G [Oic] [Nle] [BhPro][4-Cl-F] {COOH} 642 {Palm} [AC4Abu] [Aeea] [Aeea] 0.000332 0.001367 q RP [NMeArg] [Cha] [aMeS] H K G [Oic] [Nle] [BhPro] [D-BhPhe] {COOH} 615{Palm} [AC4Abu] [Aeea] [Aeea] 0.00942 0.02315 q R P [NMeArg] [Cha][BLeu] H K G [Oic] [Nle] [BhPro] [D-BhPhe] {COOH} 643 {Palm} [AC4Abu][Aeea] [Aeea] 0.000463 0.0037905 q R P [NMeArg] [Cha] [NPrG] H K G [Pip][Nle] [Aib] [4-Cl-F] {COOH} 616 {Palm} [AC4Abu] [Aeea] [Aeea] 0.0027550.01725 q R P [NMeArg] [Cha] S [Dap] K G [Oic] [Nle] [BhPro] [D-BhPhe]{COOH} 644 {Palm} [AC4Abu] [Aeea] [Aeea] 0.001397 0.004195 q R P[NMeArg] [Cha] S [NPrG] K G [Pip] [Nle] [Aib] [4-Cl-F] {COOH} 620 {Palm}[AC4Abu] [Aeea] [Aeea] 0.00742 0.01487 q R P [NMeArg] [Cha] S H [3Pal] G[Oic] [Nle] [BhPro] [D-BhPhe] {COOH} 621 {Palm} [AC4Abu] [Aeea] [Aeea]0.016415 0.0261 q R P [NMeArg] [Cha] S H [4-Cl-F] G [Oic] [Nle] [BhPro][D-BhPhe] {COOH} 622 {Palm} [AC4Abu] [Aeea] [Aeea] 0.01183 0.01965 q R P[NMeArg] [Cha] S H [4-F-F] G [Oic] [Nle] [BhPro] [D-BhPhe] {COOH} 619{Palm} [AC4Abu] [Aeea] [Aeea] 0.0262 0.125 q R P [NMeArg] [Cha] S H[aMeOrn] G [Oic] [Nle] [BhPro] [D-BhPhe] {COOH} 623 {Palm} [AC4Abu][Aeea] [Aeea] 0.001425 0.009165 q R P [NMeArg] [Cha] S H [Dab] G [Oic][Nle] [BhPro] [D-BhPhe] {COOH} 624 {Palm} [AC4Abu] [Aeea] [Aeea] 0.021050.09965 q R P [NMeArg] [Cha] S H [Dap] G [Oic] [Nle] [BhPro] [D-BhPhe]{COOH} 625 {Palm} [AC4Abu] [Aeea] [Aeea] 0.0111 0.02005 q R P [NMeArg][Cha] S H [Igl] G Oic] [Nle] [BhPro] [[D-BhPhe] {COOH} 633 {Palm}[AC4Abu] [Aeea] [Aeea] 0.0107 >4.17 [2] q R P [NMeArg] [Cha] S H [NPipG]G [Oic] [Nle] [BhPro] [D-BhPhe] {COOH} 626 {Palm} [AC4Abu] [Aeea] [Aeea]0.01217 0.02465 q R P [NMeArg] [Cha] S H K [Sar] [Oic] [Nle] [BhPro][D-BhPhe] {COOH} 613 {Palm} [AC4Abu] [Aeea] [Aeea] 0.00166 0.0388 q R P[NMeArg] [Cha] S H K G [BhPro] [Nle] [4-Cl-F] {COOH} 583 {Palm} [AC4Abu][Aeea] [Aeea] 0.005965 0.0492 q R P [NMeArg] [Cha] S H K G [Inp] [Nle][Aib] [4-Cl-F] {COOH} 582 {Palm} [AC4Abu] [Aeea] [Aeea] 0.0085 0.0293 qR P [NMeArg] [Cha] S H K G [Nip] [Nle] [Aib] [4-Cl-F] {COOH} 567 {Palm}[AC4Abu] [Aeea] [Aeea] 0.005145 0.00889 q R P [NMeArg] [Cha] S H K G[Oic] [Nle] P [D-BhPhe] {COOH} 569 {Palm} [AC4Abu] [Aeea] [Aeea] 0.0010.0009915 q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [D-BhPhe]{COOH} 570 {Palm} [AC4Abu] [Aeea] [Aeea] 0.008005 0.01975 q R P [NMeArg][Cha] S H K G [Oic] [Nle] [aMePro] [D-BhPhe] {COOH} 571 {Palm} [AC4Abu][Aeea] [Aeea] 0.001485 0.004805 q R P [NMeArg] [Cha] S H K G [Oic] [Nle][BhPro] [D-BhPhe] {COOH} 572 {Palm} [AC4Abu] [Aeea] [Aeea] 0.0010960.00768 q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [bAla] [D-BhPhe] {COOH}573 {Palm} [AC4Abu] [Aeea] [Aeea] 0.002165 0.004245 q R P [NMeArg] [Cha]S H K G [Oic] [Nle] [D-BhPhe] {COOH} 575 {Palm} [AC4Abu] [Aeea] [Aeea]0.003585 0.014095 q R P [NMeArg] [Cha] S H K G [Oic] [Nle] P [BhPhe]{COOH} 577 {Palm} [AC4Abu] [Aeea] [Aeea] 0.001775 0.004425 q R P[NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [BhPhe] {COOH} 578 {Palm}[AC4Abu] [Aeea] [Aeea] 0.001805 0.004895 q R P [NMeArg] [Cha] S H K G[Oic] [Nle] [BhPhe] {COOH} 580 {Palm} [AC4Abu] [Aeea] [Aeea] 0.00046150.0007365 q R P [NMeArg] [Cha] S H K G [Oic] [Aib] [4-Cl-F] {COOH} 584{Palm} [AC4Abu] [Aeea] [Aeea] 0.0229 0.0522 q R P [NMeArg] [Cha] S H K G[Oic] [Nle] [1-Ach] [4-Cl-F] {COOH} 585 {Palm} [AC4Abu] [Aeea] [Aeea]0.006735 0.0223 q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Acp] [4-Cl-F]{COOH} 586 {Palm} [AC4Abu] [Aeea] [Aeea] 0.007965 0.02295 q R P [NMeArg][Cha] S H K G [Oic] [Nle] [Nip] [4-Cl-F] {COOH} 587 {Palm} [AC4Abu][Aeea] [Aeea] 0.001292 0.003585 q R P [NMeArg] [Cha] S H K G [Oic] [Nle][Inp] [4-Cl-F] {COOH} 600 {Palm} [AC4Abu] [Aeea] [Aeea] 0.0016550.004455 q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [BhPro] [D-4ClF]{COOH} 601 {Palm} [AC4Abu] [Aeea] [Aeea] 0.003435 0.003255 q R P[NMeArg] [Cha] S H K G [Oic] [Nle] [Pip] [D-4ClF] {COOH} 602 {Palm}[AC4Abu] [Aeea] [Aeea] 0.00548 0.02565 q R P [NMeArg] [Cha] S H K G[Oic] [Nle] [Nip] [D-4ClF] {COOH} 603 {Palm} [AC4Abu] [Aeea] [Aeea]0.002955 0.03235 q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Inp] [D-4ClF]{COOH} 604 {Palm} [AC4Abu] [Aeea] [Aeea] 0.0004225 0.000512 q R P[NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [D-4ClF] {COOH} 606 {Palm}[AC4Abu] [Aeea] [Aeea] 0.000307 0.00137 q R P [NMeArg] [Cha] S H K G[Oic] [Nle] P [AMEF] {COOH} 607 {Palm} [AC4Abu] [Aeea] [Aeea] 0.000095650.0005505 q R P [NMeArg] [Cha] S H K G [Oic] [Nle] P [D-AMF] {COOH} 608{Palm} [AC4Abu] [Aeea] [Aeea] 0.0002135 0.0003255 q R P [NMeArg] [Cha] SH K G [Oic] [Nle] [Aib] [AMEF] {COOH} 609 {Palm} [AC4Abu] [Aeea] [Aeea]0.001245 0.002495 q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Pip] [AMEF]{COOH} 610 {Palm} [AC4Abu] [Aeea] [Aeea] 0.0010645 0.004985 q R P[NMeArg] [Cha] S H K G [Oic] [Nle] [aMePro] [AMEF] {COOH} 611 {Palm}[AC4Abu] [Aeea] [Aeea] 0.000722 0.0015685 q R P [NMeArg] [Cha] S H K G[Oic] [Nle] [BhPro] [AMEF] {COOH} 612 {Palm} [AC4Abu] [Aeea] [Aeea]0.00172 0.004625 q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [BhPro][D-AMF] {COOH} 617 {Palm} [AC4Abu] [Aeea] [Aeea] 0.008675 0.0258 q R P[NMeArg] [Cha] S h K G [Oic] [Nle] [BhPro] [D-BhPhe] {COOH} 568 {Palm}[AC4Abu] [Aeea] [Aeea] 0.002965 0.004545 q R P [NMeArg] [Cha] S H K G[Pip] [Nle] P [D-BhPhe] {COOH} 574 {Palm} [AC4Abu] [Aeea] [Aeea]0.0006525 0.00283 q R P [NMeArg] [Cha] S H K G [Pip] [Nle] [D-BhPhe]{COOH} 579 {Palm} [AC4Abu] [Aeea] [Aeea] 0.000831 0.002505 q R P[NMeArg] [Cha] S H K G [Pip] [Nle] [BhPhe] {COOH} 581 {Palm} [AC4Abu][Aeea] [Aeea] 0.001401 0.00585 q R P [NMeArg] [Cha] S H K G [Pip] [Aib][4-Cl-F] {COOH} 588 {Palm} [AC4Abu] [Aeea] [Aeea] 0.00152 0.00637 q R P[NMeArg] [Cha] S H K G [Pip] [Nle] [Nip] [4-Cl-F] {COOH} 589 {Palm}[AC4Abu] [Aeea] [Aeea] 0.000624 0.00415 Q R P [NMeArg] [Cha] S H K G[Pip] [Nle] [Inp] [4-Cl-F] {COOH} 590 {Palm} [AC4Abu] [Aeea] [Aeea]0.000816 0.0062 q R P [NMeArg] [Cha] S H K G [Pip] [Nle] P [Aic] {COOH}597 {Palm} [AC4Abu] [Aeea] 0.000885 0.00337 [Aeea] q R P [NMeArg] [Cha]S H K G [Pip] [Nle] [Aic] {COOH} 598 {Palm} [AC4Abu] [Aeea] 0.0004630.002845 [Aeea] q R P [NMeArg] [Cha] S H K G [Pip] [Nle] P [Tic] {COOH}599 {Palm} [AC4Abu] [Aeea] 0.00044 0.001012 [Aeea] q R P [NMeArg] [Cha]S H K G [Pip] [Nle] P [D-Tic] {COOH} 647 {Palm} [AC4Abu] [Aeea] 0.0015790.00583 [Aeea] q R P [NMeArg] [Cha] S H K G [Pip] [Nle] P [BhPhe] {COOH}618 {Palm} [AC4Abu] [Aeea] [Aeea] 0.002465 0.0111 q R P [NMeArg] [Cha] SH O G [Oic] [Nle] [BhPro] [D-BhPhe] {COOH} 593 {Palm} [AC4Abu] [Aeea][Aeea] 0.0001465 0.000231 Q R P [NMeArg] L S H K G P [Nle] P [4-Cl-F]{COOH} 645 {Palm} [AC4Abu] [Aeea] 0.00025385 0.0003945 [Aeea] q R P[NPipG] [Cha] S H K G [Pip] [Nle] [Aib] [4-Cl-F] {COOH} 495 {PDA}[AC4Abu] [Aeea] [Aeea] 0.00008295 0.0005325 Q R P [NMeArg] L S H K G P[Nle] P [4-Cl-F] {COOH} 118 {pIFBu} [AC4Abu] [Aeea] [Aeea] 0.000047550.0001715 Q R P [NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 474{Sebacicacid} [AC4Abu] 0.0011385 0.00376 [Aeea] [Aeea] [hArg] Q [hArg] P[NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 291 {Sebacicacid}[AC4Abu] [Aeea] 0.003375 0.00247 [Aeea] [hArg] r Q [hArg] P r [NMeLeu] SH K G [Oic] [pI-Phe] P [D-Bip] {COOH} 289 {Sebacicacid} [AC4Abu] [Aeea]0.000914 0.0013 [Aeea] [hArg] r Q [hArg] P r [NMeLeu] S H K G [Oic][pI-Phe] P [D-Bip] {COOH} 288 {Sebacicacid} [AC4Abu] [Aeea] 0.038550.0385 [Aeea] Q [hArg] P r [NMeLeu] S H K G [Oic] [pI-Phe] P [D-Bip]{COOH} 509 {Sebacicacid} [AC4Abu] [Aeea] 0.000179 0.0008645 [Aeea] Q R P[NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 594 {St} [AC4Abu] [Aeea][Aeea] 0.0002455 0.000528 Q R P [NMeArg] L S H K G P [Nle] P [4-Cl-F]{COOH} 508 {Subericacid} [AC4Abu] [Aeea] 0.0000768 0.0004665 [Aeea] Q RP [NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 497 {Succinicacid}[AC4Abu] [Aeea] 0.000208 0.0013918 [Aeea] Q R P [NMeArg] L S H K G P[Nle] P [4-Cl-F] {COOH} 534 {IDA} [AC4Abu] [AC4Abu] 0.000683 0.00375[Aeea] [Aeea] q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl-F]{COOH} 399 {IDA} [AC4Abu] [AC4Abu] 0.0000335 0.0001044 [Aeea] [Aeea] Q RP [NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 535 {IDA} [AC4Abu][AC4Abu] 0.000798 0.005145 [Aeea] q R P [NMeArg] [Cha] S H K G [Oic][Nle] [Aib] [4-Cl-F] {COOH} 462 {IDA} [AC4Abu] [AC4Abu] 0.00001540.00007925 [Aeea] Q R P [NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 533{TDA} [AC4Abu] [AC4Abu] q R 0.000629 0.003875 P [NMeArg] [Cha] S H K G[Oic] [Nle] [Aib] [4-Cl-F] {COOH} 536 {IDA} [AC4Abu] [Aeea] 0.0005920.00203 [AC4Abu] q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl-F]{COOH} 398 {IDA} [AC4Abu] [Aeea] [Aeea] 0.00005735 0.0001163 [Aeea][Aeea] [Aeea] [Aeea] Q R P [NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH}375 {IDA} [AC4Abu] [Aeea] [Aeea] 0.00015535 0.000288 [aMeLeu] R P[NMeArg] [aMeLeu] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 299 {IDA}[AC4Abu] [Aeea] [Aeea] 0.02435 0.0146 [BhAsn] [hArg] P [NMeArg] [Cha] SH K G [Oic] [Nle] P [D-4ClF] {COOH} 420 {IDA} [AC4Abu] [Aeea] [Aeea]0.00005255 0.0000838 [BhAsn] R P [NMeArg] L S H K G P [Nle] P [4-Cl-F]{COOH} 463 {IDA} [AC4Abu] [Aeea] [Aeea] 0.00002005 0.0001275 [BhGln] R P[NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 300 {IDA} [AC4Abu] [Aeea][Aeea] 0.0415 0.03635 [BhLeu] [hArg] P [NMeArg] [Cha] S H K G [Oic][Nle] P [D-4ClF] {COOH} 421 {IDA} [AC4Abu] [Aeea] [Aeea] 0.00009340.0002855 [BhLeu] R P [NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 298{IDA} [AC4Abu] [Aeea] [Aeea] 0.03285 0.0249 [BLeu] [hArg] P [NMeArg][Cha] S H K G [Oic] [Nle] P [D-4ClF] {COOH} 552 {TDA} [AC4Abu] [Aeea][Aeea] 0.001955 0.0048775 [BLeu] R [aMePro] [hArg] [aMeLeu] S H K G[Oic] [Nle] [aMePro] [4-Cl-F] {COOH} 395 {TDA} [AC4Abu] [Aeea] [Aeea]0.000741 0.00102 [BLeu] R P [hArg] [BLeu] S H K G [Oic] [Nle] P [4-Cl-F]{COOH} 374 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00030537 0.00050275 [BLeu] R P[NMeArg] [aMeLeu] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 388 {TDA}[AC4Abu] [Aeea] [Aeea] 0.000706 0.0007275 [BLeu] R P [NMeArg] [aMeLeu] SH K G [Oic] [Nle] [aMePro] [4-Cl-F] {COOH} 394 {TDA} [AC4Abu] [Aeea][Aeea] 0.000297 0.001498 [BLeu] R P [NMeArg] [BLeu] S H K G [Oic] [Nle]P [4-Cl-F] {COOH} 393 {TDA} [AC4Abu] [Aeea] [Aeea] 0.000769 0.002665[BLeu] R P [NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 558{TDA} [AC4Abu] [Aeea] 0.0034 0.01249 [Aeea] [Deg] [hArg] P r [Cha] S H KG [Oic] [Nle] P [4-Cl-F] {COOH} 443 {TDA} [AC4Abu] [Aeea] [Aeea] 0.002310.00167 [Deg] R P [NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH}309 {TDA} [AC4Abu] [Aeea] [Aeea] 0.000181 0.0002155 [hArg] [hArg] Q[hArg] P [NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 321 {TDA}[AC4Abu] [Aeea] [Aeea] 0.0179 0.02905 [hArg] [Nle] Q [hArg] P r [NMeLeu]A H K G [Oic] [pI-Phe] P [D-Bip] {COOH} 333 {TDA} [AC4Abu] [Aeea] 0.03610.04665 [Aeea] [hArg] [Nle] Q [hArg] P r [NMeLeu] A A K G [Oic] [pI-Phe]P [D-Bip] {COOH} 315 {TDA} [AC4Abu] [Aeea] [Aeea] 0.003045 0.0049 [hArg][Nle] Q [hArg] P r [NMeLeu] S H K G [Oic] [pI-Phe] P [D-Bip] {COOH} 327{TDA} [AC4Abu] [Aeea] [Aeea] 0.014445 0.014805 [hArg] [Nle] Q [hArg] P r[NMeLeu] S A K G [Oic] [pI-Phe] P [D-Bip] {COOH} 490 {TDA} [AC4Abu][Aeea] [Aeea] 0.00002765 0.0001015 [hArg] Q [hArg] P [hArg] [Cha] S H KG [Oic] [Nle] P [4-Cl-F] {COOH} 310 {TDA} [AC4Abu] [Aeea] [Aeea]0.000226 0.0003635 [hArg] Q [hArg] P [NMeArg] [Cha] S H K G [Oic] [Nle]P [4-Cl-F] {COOH} 317 {TDA} [AC4Abu] [Aeea] [Aeea] 0.05685 0.0639 [hArg]r Q [hArg] P [Nle] [NMeLeu] A H K G [Oic] [pI-Phe] P [D-Bip] {COOH} 329{TDA} [AC4Abu] [Aeea] [Aeea] 0.03755 0.03615 [hArg] r Q [hArg] P [Nle][NMeLeu] A A K G [Oic] [pI-Phe] P [D-Bip] {COOH} 311 {TDA} [AC4Abu][Aeea] [Aeea] 0.0347 0.03735 [hArg] r Q [hArg] P [Nle] [NMeLeu] S H K G[Oic] [pI-Phe] P [D-Bip] {COOH} 323 {TDA} [AC4Abu] [Aeea] [Aeea]0.0025055 0.005295 [hArg] r Q [hArg] P [Nle] [NMeLeu] S A K G [Oic][pI-Phe] P [D-Bip] {COOH} 331 {TDA} [AC4Abu] [Aeea] [Aeea] 0.02470.01805 [hArg] r Q [hArg] P K [NMeLeu] A A [Nle] G [Oic] [pI-Phe] P[D-Bip] {COOH} 319 {TDA} [AC4Abu] [Aeea] [Aeea] 0.07325 0.0631 [hArg] rQ [hArg] P K [NMeLeu] A H [Nle] G [Oic] [pI-Phe] P [D-Bip] {COOH} 325{TDA} [AC4Abu] [Aeea] [Aeea] 0.003955 0.00476 [hArg] r Q [hArg] P K[NMeLeu] S A [Nle] G [Oic] [pI-Phe] P [D-Bip] {COOH} 313 {TDA} [AC4Abu][Aeea] [Aeea] 0.01475 0.010445 [hArg] r Q [hArg] P K [NMeLeu] S H [Nle]G [Oic] [pI-Phe] P [D-Bip] {COOH} 330 {TDA} [AC4Abu] [Aeea] [Aeea]0.0397 0.05605 [hArg] r Q [hArg] P r [NMeLeu] A A [Nle] G [Oic] [pI-Phe]P [D-Bip] {COOH} 318 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00744 0.04595 [hArg]r Q [hArg] P r [NMeLeu] A H [Nle] G [Oic] [pI-Phe] P [D-Bip] {COOH} 324{TDA} [AC4Abu] [Aeea] [Aeea] 0.014865 0.02035 [hArg] r Q [hArg] P r[NMeLeu] S A [Nle] G [Oic] [pI-Phe] P [D-Bip] {COOH} 312 {TDA} [AC4Abu][Aeea] [Aeea] 0.013575 0.01063 [hArg] r Q [hArg] P r [NMeLeu] S H [Nle]G [Oic] [pI-Phe] P [D-Bip] {COOH} 334 {TDA} [AC4Abu] [Aeea] [Aeea]0.03415 0.0447 [hArg] r Q [Nle] P r [NMeLeu] A A K G [Oic] [pI-Phe] P[D-Bip] {COOH} 322 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0163 0.02645 [hArg] rQ [Nle] P r [NMeLeu] A H K G [Oic] [pI-Phe] P [D-Bip] {COOH} 328 {IDA}[AC4Abu] [Aeea] [Aeea] 0.00917 0.0077 [hArg] r Q [Nle] P r [NMeLeu] S AK G [Oic] [pI-Phe] P [D-Bip] {COOH} 316 {IDA} [AC4Abu] [Aeea] [Aeea]0.006005 0.009295 [hArg] r Q [Nle] P r [NMeLeu] S H K G [Oic] [pI-Phe] P[D-Bip] {COOH} 347 {IDA} [AC4Abu] [Aeea] [Aeea] 0.001885 0.00213 [Nle] QR P [hArg] [Cha] A A K G [Oic] [Nle] P [4-Cl-F] {COOH} 342 {IDA}[AC4Abu] [Aeea] [Aeea] 0.0008035 0.0006845 [Nle] Q R P [hArg] [Cha] S AK G [Oic] [Nle] P [4-Cl-F] {COOH} 338 {IDA} [AC4Abu] [Aeea] [Aeea]0.00056 0.000766 [Nle] Q R P [hArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F]{COOH} 332 {IDA} [AC4Abu] [Aeea] [Aeea] 0.03995 0.05055 [Nle] r Q [hArg]P r [NMeLeu] A A K G [Oic] [pI-Phe] P [D-Bip] {COOH} 320 {IDA} [AC4Abu][Aeea] [Aeea] 0.01785 0.03765 [Nle] r Q [hArg] P r [NMeLeu] A H K G[Oic] [pI-Phe] P [D-Bip] {COOH} 326 {IDA} [AC4Abu] [Aeea] [Aeea]0.014765 0.01777 [Nle] r Q [hArg] P r [NMeLeu] S A K G [Oic] [pI-Phe] P[D-Bip] {COOH} 314 {IDA} [AC4Abu] [Aeea] [Aeea] 0.00277 0.006815 [Nle] rQ [hArg] P r [NMeLeu] S H K G [Oic] [pI-Phe] P [D-Bip] {COOH} 557 {IDA}[AC4Abu] [Aeea] [Aeea] 0.00612 0.010425 [Tle] [hArg] P r [Cha] S H K G[Oic] [Nle] P [4-Cl-F] {COOH} 442 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0020050.00216 [Tle] R P [NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH}292 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00578 0.006675 1 [hArg] P [NMeArg][Cha] S H K G [Oic] [Nle] P [D-4ClF] {COOH} 307 {TDA} [AC4Abu] [Aeea][Aeea] 0.000305 0.0003105 O F [hArg] [hArg] [BLeu] [hArg] P [hArg] [Cha]S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 297 {TDA} [AC4Abu] [Aeea] [Aeea]0.0006975 0.0010165 O F [hArg] [hArg] Q [NMeArg] P [NMeArg] [Cha] S H KG [Oic] [Nle] P [4-Cl-F] {COOH} 550 {TDA} [AC4Abu] [Aeea] [Aeea] 0.09480.13525 Q [BhPro] [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl-F]{COOH} 400 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00009275 0.00042 Q [Cit] P[NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 454 {TDA} [AC4Abu] [Aeea][Aeea] 0.001072 0.00535 q [hArg] [Aib] r [Cha] S H K G [Oic] [Nle] P[4-Cl-F] {COOH} 301 {TDA} [AC4Abu] [Aeea] [Aeea] 0.03595 0.029 q [hArg][BhPro] [NMeArg] [Cha] S H K G [Oic] [Nle] P [D-4ClF] {COOH} 455 {TDA}[AC4Abu] [Aeea] [Aeea] 0.002475 0.010265 q [hArg] [Deg] r [Cha] S H K G[Oic] [Nle] P [4-Cl-F] {COOH} 486 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0017950.00404 q [hArg] [Oic] r [Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 488{TDA} [AC4Abu] [Aeea] [Aeea] 0.0001975 0.001725 q [hArg] P [hArg] [Cha]S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 489 {TDA} [AC4Abu] [Aeea] [Aeea]0.00060125 0.00359 Q [hArg] P [hArg] [Cha] S H K G [Oic] [Nle] P[4-Cl-F] {COOH} 358 {TDA} [AC4Abu] [Aeea] [Aeea] 0.000285 0.00031433 Q[hArg] P [hArg] L S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 357 {TDA}[AC4Abu] [Aeea] [Aeea] 0.000073 0.00023967 Q [hArg] P [hArg] L S H K G P[Nle] P [4-Cl-F] {COOH} 359 {TDA} [AC4Abu] [Aeea] [Aeea] 0.000052950.000116 Q [hArg] P [NMeArg] [aMeLeu] S H K G [Oic] [Nle] P [4-Cl-F]{COOH} 460 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0000347 0.00006275 Q [hArg] P[NMeArg] [aMeLeu] S H K G P [Nle] P [4-Cl-F] {COOH} 473 {TDA} [AC4Abu][Aeea] [Aeea] 0.00009905 0.000465 Q [hArg] P [NMeArg] [aMeLeu] S H K G P[Nle] P [2Nal] {COOH} 302 {TDA} [AC4Abu] [Aeea] [Aeea] 0.016495 0.01435q [hArg] P [NMeArg] [BhLeu] S H K G [Oic] [Nle] P [D-4ClF] {COOH} 383{TDA} [AC4Abu] [Aeea] [Aeea] 0.000535 0.0013695 Q [hArg] P [NMeArg][BLeu] S H K G P [Nle] P [4-Cl-F] {COOH} 303 {TDA} [AC4Abu] [Aeea][Aeea] 0.0792 0.07415 q [hArg] P [NMeArg] [Cha] [bAla] H K G [Oic] [Nle]P [D-4ClF] {COOH} 304 {TDA} [AC4Abu] [Aeea] [Aeea] 0.1394 0.2605 q[hArg] P [NMeArg] [Cha] S [bAla] K G [Oic] [Nle] P [D-4ClF] {COOH} 305{TDA} [AC4Abu] [Aeea] [Aeea] 0.078 0.06665 q [hArg] P [NMeArg] [Cha] S H[3Pal] G [Oic] [Nle] P [D-4ClF] {COOH} 293 {TDA} [AC4Abu] [Aeea] [Aeea]0.10915 0.11 q [hArg] P [NMeArg] [Cha] S H [bAla] G [Oic] [Nle] P[D-4ClF] {COOH} 306 {TDA} [AC4Abu] [Aeea] [Aeea] 0.07405 0.1365 q [hArg]P [NMeArg] [Cha] S H [BLys] G [Oic] [Nle] P [D-4ClF] {COOH} 294 {TDA}[AC4Abu] [Aeea] [Aeea] >4.17 [2] 0.0307 q [hArg] P [NMeArg] [Cha] S H K[bAla] [Oic] [Nle] P [D-4ClF] {COOH} 295 {TDA} [AC4Abu] [Aeea] [Aeea]0.00329 0.01039 q [hArg] P [NMeArg] [Cha] S H K G [Oic] [Nle] [bAla][D-4ClF] {COOH} 296 {TDA} [AC4Abu] [Aeea] [Aeea] 0.002745 0.00232 q[hArg] P [NMeArg] [Cha] S H K G [Oic] [Nle] [BhPro] [D-4ClF] {COOH} 362{TDA} [AC4Abu] [Aeea] [Aeea] 0.00197 0.0037522 Q [hArg] P [NMeArg] [Cha]S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 405 {TDA} [AC4Abu] [Aeea] [Aeea]0.005755 0.013845 Q [hArg] P [NMeArg] [Cha] S H K G [Oic] [Nle] P [2Nal]{COOH} 478 {TDA} [AC4Abu] [Aeea] [Aeea] 0.000496 0.0008075 q [hArg] P[NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 408 {TDA} [AC4Abu][Aeea] [Aeea] 0.0000768 0.0001841 Q [hArg] P [NMeArg] [Cha] S H K G P[Nle] P [4-Cl-F] {COOH} 354 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0002790.0003615 Q [hArg] P [NMeArg] L S H K G [Oic] [Nle] P [4-Cl-F] {COOH}353 {TDA} [AC4Abu] [Aeea] [Aeea] 0.000095 0.00027467 Q [hArg] P [NMeArg]L S H K G P [Nle] P [4-Cl-F] {COOH} 483 {TDA} [AC4Abu] [Aeea] [Aeea]0.0000754 0.000229 Q [hArg] P [NMehArg] L S H K G P [Nle] P [4-Cl-F]{COOH} 451 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0112 0.03165 q [hArg] P r[Cha] S [Deg] K G [Oic] [Nle] P [4-Cl-F] {COOH} 452 {TDA} [AC4Abu][Aeea] [Aeea] 0.01044 0.025 q [hArg] P r [Cha] S [Tle] K G [Oic] [Nle] P[4-Cl-F] {COOH} 372 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00284 0.001495 q[hArg] P r [Cha] S A K G [Oic] [Nle] P [4-Cl-F] {COOH} 371 {TDA}[AC4Abu] [Aeea] [Aeea] 0.00692 0.00467 q [hArg] P r [Cha] S H K [Tle][Oic] [Nle] P [4-Cl-F] {COOH} 363 {TDA} [AC4Abu] [Aeea] [Aeea] 0.003270.003955 q [hArg] P r [Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 364{TDA} [AC4Abu] [Aeea] [Aeea] 0.006 0.02035 Q [hArg] P r [Cha] S H K G[Oic] [Nle] P [D-4ClF] {COOH} 365 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0230.02065 q [hArg] P r [Cha] S H K G [Oic] [Nle] P [Oic] {COOH} 366 {TDA}[AC4Abu] [Aeea] [Aeea] 0.01715 0.0236 q [hArg] P r [Cha] S H K G [Oic][Nle] P [Aic] {COOH} 367 {TDA} [AC4Abu] [Aeea] [Aeea] 0.02635 0.0675 q[hArg] P r [Cha] S H K G [Oic] [Nle] P [1-Nal] {COOH} 368 {TDA} [AC4Abu][Aeea] [Aeea] 0.0323 0.03135 q [hArg] P r [Cha] S H K G [Oic] [Nle] P[2-Nal] {COOH} 369 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0259 0.04205 q [hArg]P r [Cha] S H K G [Oic] [Nle] P [D-4IF] {COOH} 370 {TDA} [AC4Abu] [Aeea][Aeea] 0.01291 0.0237 q [hArg] P r [Cha] S H K G [Oic] [Nle] P [B114ClF]{COOH} 406 {TDA} [AC4Abu] [Aeea] [Aeea] 0.03355 0.07295 q [hArg] P r[Cha] S H K G [Oic] [Nle] P [CPG] {COOH} 407 {TDA} [AC4Abu] [Aeea][Aeea] 0.12395 0.216 q [hArg] P r [Cha] S H K G [Oic] [Nle] P [D-2Nal]{COOH} 431 {TDA} [AC4Abu] [Aeea] [Aeea] 0.03125 0.025 q [hArg] P r [Cha]S H K G [Oic] [Nle] P [D-Igl] {COOH} 432 {TDA} [AC4Abu] [Aeea] [Aeea]0.01105 0.01615 q [hArg] P r [Cha] S H K G [Oic] [Nle] P [B4ClF] {COOH}453 {TDA} [AC4Abu] [Aeea] [Aeea] 0.01895 0.0208 q [hArg] P r [Cha] S H KG [Oic] [Nle] [D-4IF] {COOH} 595 {TDA} [AC4Abu] [Aeea] [Aeea] 0.050050.05475 q [hArg] P r [Cha] S H K G [Oic] [Nle] [Aic] {COOH} 596 {TDA}[AC4Abu] [Aeea] [Aeea] 0.005135 0.0339 q [hArg] P r [Cha] S H K G [Oic][Nle] [Oic] {COOH} 373 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00281 0.003315 q[hArg] P r [Cha] S Y K G [Oic] [Nle] P [4-Cl-F] {COOH} 434 {TDA}[AC4Abu] [Aeea] [Aeea] 0.010995 0.011405 q [hArg] P r [Deg] S H K G[Oic] [Nle] P [4-Cl-F] {COOH} 433 {TDA} [AC4Abu] [Aeea] [Aeea] 0.012850.01975 q [hArg] P r [Tle] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 397{TDA} [AC4Abu] [Aeea] [Aeea] 0.00636 0.016515 Q [NMeArg] [aMeLeu] S H KG [Oic] [Nle] P [4-Cl-F] {COOH} 484 {TDA} [AC4Abu] [Aeea] [Aeea]0.000176 0.000767 Q [NMehArg] P [NMeArg] [aMeLeu] S H K G P [Nle] P[4-Cl-F] {COOH} 524 {TDA} [AC4Abu] [Aeea] [Aeea] 0.05315 0.346 q E P[NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl-F] {COOH} 525 {TDA}[AC4Abu] [Aeea] [Aeea] 0.1585 0.243 q e P [NMeArg] [Cha] S H K G [Oic][Nle] [Aib] [4-Cl-F] {COOH} 475 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0103450.03585 Q E P [NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 520 {TDA}[AC4Abu] [Aeea] [Aeea] 0.009175 0.05615 Q P [NMeArg] [Cha] S H K G [Oic][Nle] [Aib] [4-Cl-F] {COOH} 482 {TDA} [AC4Abu] [Aeea] [Aeea] 0.000086250.00020283 Q R [aMePro] [hArg] [aMeLeu] S H K G P [Nle] P [4-Cl-F]{COOH} 392 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0005845 0.0008555 Q R [aMePro][hArg] [BLeu] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 501 {TDA} [AC4Abu][Aeea] [Aeea] 0.010927 0.068175 q R [aMePro] [hArg] [BLeu] S H K G [Pip][Nle] [4-Cl-F] {COOH} 502 {TDA} [AC4Abu] [Aeea] [Aeea] 0.000456250.004815 q R [aMePro] [hArg] [BLeu] S H K G [Oic] [Nle] [aMePro][4-Cl-F] {COOH} 503 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00016 0.002125 q R[aMePro] [hArg] [BLeu] S H K G [Pip] [Nle] [aMePro] [4-Cl-F] {COOH} 504{TDA} [AC4Abu] [Aeea] [Aeea] 0.001394 0.023075 q R [aMePro] [hArg][BLeu] S H K G [aMePro] [Nle] [aMePro] [4-Cl-F] {COOH} 505 {TDA}[AC4Abu] [Aeea] [Aeea] 0.00007075 0.000516 q R [aMePro] [hArg] [BLeu] SH K G [Oic] [Nle] [Aib] [4-Cl-F] {COOH} 519 {TDA} [AC4Abu] [Aeea] [Aeea]0.009735 0.0447 q R [aMePro] [hArg] [BLeu] S H K G [Oic] [Nle] [4-Cl-F]{COOH} 556 {TDA} [AC4Abu] [Aeea] [Aeea] 0.018 0.0691 q R [aMePro] [hArg][BLeu] S H K G [Oic] [Nle] [BhPro] [4-Cl-F] {COOH} 523 {TDA} [AC4Abu][Aeea] [Aeea] 0.02565 0.0719 q R [aMePro] [Nle] [BLeu] S H K G [Oic][Nle] [aMePro] [4-Cl-F] {COOH} 522 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0005230.006765 q R [BhPro] [hArg] [BLeu] S H K G [Oic] [Nle] [Aib] [4-Cl-F]{COOH} 553 {TDA} [AC4Abu] [Aeea] [Aeea] 0.002227 0.00201 q R [BhPro][NMeArg] [aMeLeu] S H K G [Oic] [Nle] [aMePro] [4-Cl-F] {COOH} 441 {TDA}[AC4Abu] [Aeea] [Aeea] 0.001895 0.001815 Q R [BhPro] [NMeArg] [Cha] S HK G [Oic] [Nle] P [4-Cl-F] {COOH} 516 {TDA} [AC4Abu] [Aeea] [Aeea]0.000557 0.0020225 q R [BhPro] [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib][4-Cl-F] {COOH} 396 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0009765 0.00114 Q R[NMeArg] [aMeLeu] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 447 {TDA}[AC4Abu] [Aeea] [Aeea] 0.00966 0.014805 Q R [NMeArg] [BLeu] S H K G[Oic] [Nle] P [4-Cl-F] {COOH} 446 {TDA} [AC4Abu] [Aeea] 0.00111050.001895 [Aeea] Q R [NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH}521 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0010255 0.0032075 q R [Pip] [hArg][BLeu] S H K G [Oic] [Nle] [Aib] [4-Cl-F] {COOH} 440 {TDA} [AC4Abu][Aeea] [Aeea] 0.003485 0.004265 Q R [Pip] [NMeArg] [Cha] S H K G [Oic][Nle] P [4-Cl-F] {COOH} 517 {TDA} [AC4Abu] [Aeea] [Aeea] 0.000620.0032225 q R [Pip] [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl-F]{COOH} 476 {TDA} [AC4Abu] [Aeea] [Aeea] 0.000346 0.0008725 Q R E[NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 360 {TDA} [AC4Abu] [Aeea][Aeea] 0.0000519 0.000082733 Q R P [hArg] [aMeLeu] S H K G [Oic] [Nle] P[4-Cl-F] {COOH} 404 {TDA} [AC4Abu] [Aeea] [Aeea] 0.000298 0.0003615 Q RP [hArg] [aMeLeu] S H K G P [Nle] P [2Nal] {COOH} 485 {TDA} [AC4Abu][Aeea] [Aeea] 0.00011305 0.00041 Q r P [hArg] [aMeLeu] S H K G P [Nle] P[4-Cl-F] {COOH} 378 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00025955 0.0004115 qR P [hArg] [BLeu] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 308 {TDA}[AC4Abu] [Aeea] [Aeea] 0.00008665 0.00018 Q R P [hArg] [Cha] S H K G[Oic] [Nle] P [4-Cl-F] {COOH} 356 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00032050.00028933 Q R P [hArg] L S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 355{TDA} [AC4Abu] [Aeea] [Aeea] 0.00002445 0.00006 Q R P [hArg] L S H K G P[Nle] P [4-Cl-F] {COOH} 529 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00526 0.024 qR P [Nle] [Cha] S A K G [Oic] [Nle] [Aib] [4-Cl-F] {COOH} 528 {TDA}[AC4Abu] [Aeea] [Aeea] 0.0056325 0.021825 q R P [Nle] [Cha] S H K G[Oic] [Nle] [Aib] [4-Cl-F] {COOH} 428 {TDA} [AC4Abu] [Aeea] [Aeea]0.0004005 0.000494 Q R P [Nle] L S H K G P [Nle] P [4-Cl-F] {COOH} 518{TDA} [AC4Abu] [Aeea] [Aeea] 0.00011295 0.000444 q R P [NMeArg] [aMeLeu][BhSer] H K G [Oic] [Nle] [aMePro] [4-Cl-F] {COOH} 387 {TDA} [AC4Abu][Aeea] [Aeea] 0.00010873 0.00016625 Q R P [NMeArg] [aMeLeu] S H K G[aMePro] [Nle] P [4-Cl-F] {COOH} 376 {TDA} [AC4Abu] [Aeea] [Aeea]0.0002375 0.0004145 Q R P [NMeArg] [aMeLeu] S H K G [Oic] [Nle] [aMePro][4-Cl-F] {COOH} 385 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00006875 0.0001705 QR P [NMeArg] [aMeLeu] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 386 {TDA}[AC4Abu] [Aeea] [Aeea] 0.0000934 0.00026113 q R P [NMeArg] [aMeLeu] S HK G [Oic] [Nle] P [4-Cl-F] {COOH} 389 {TDA} [AC4Abu] [Aeea] [Aeea]0.00019648 0.00036525 Q R P [NMeArg] [aMeLeu] S H K G [Oic] [Nle][4-Cl-F] {COOH} 554 {TDA} [AC4Abu] [Aeea] [Aeea] 0.000867 0.0028325 q RP [NMeArg] [aMeLeu] S H K G [Oic] [Nle] [BhPro] [4-Cl-F] {COOH} 555{TDA} [AC4Abu] [Aeea] [Aeea] 0.0001024 0.0004215 q R P [NMeArg] [aMeLeu]S H K G [Pip] [Nle] [aMePro] [4-Cl-F] {COOH} 605 {TDA} [AC4Abu] [Aeea][Aeea] 0.00008315 0.000298 Q R P [NMeArg] [aMeLeu] S H K G [Pip] [Nle][aMePro] [4-Cl-F] {COOH} 457 {TDA} [AC4Abu] [Aeea] [Aeea] 0.000022950.0000564 Q R P [NMeArg] [aMeLeu] S H K G P [Nle] P [4-Cl-F] {COOH} 472{TDA} [AC4Abu] [Aeea] [Aeea] 0.00002035 0.000092 Q R P [NMeArg] [aMeLeu]S H K G P [Nle] P [2Nal] {COOH} 422 {TDA} [AC4Abu] [Aeea] [Aeea]0.00016875 0.0001965 Q R P [NMeArg] [BhLeu] S H K G P [Nle] P [4-Cl-F]{COOH} 391 {TDA} [AC4Abu] [Aeea] [Aeea] 0.000565 0.001503 Q R P [NMeArg][BLeu] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 458 {TDA} [AC4Abu] [Aeea][Aeea] 0.00002425 0.0001195 Q R P [NMeArg] [BLeu] S H K G P [Nle] P[4-Cl-F] {COOH} 500 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0005875 0.0031378 q RP [NMeArg] [Cha] [BhSer] H K G [Oic] [Nle] [Aib] [4-Cl-F] {COOH} 438{TDA} [AC4Abu] [Aeea] [Aeea] 0.00149 0.0012775 Q R P [NMeArg] [Cha] S[Tle] K G [Oic] [Nle] P [4-Cl-F] {COOH} 419 {TDA} [AC4Abu] [Aeea] [Aeea]0.0004845 0.000379 Q R P [NMeArg] [Cha] S A K G [Oic] [Nle] P [4-Cl-F]{COOH} 530 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00037975 0.0009745 q R P[NMeArg] [Cha] S A K G [Oic] [Nle] [Aib] [4-Cl-F] {COOH} 444 {TDA}[AC4Abu] [Aeea] [Aeea] 0.0056 0.01006 Q R P [NMeArg] [Cha] S H [AMe-K] G[Oic] [Nle] P [4-Cl-F] {COOH} 543 {TDA} [AC4Abu] [Aeea] [Aeea]0.00058275 0.0014035 q R P [NMeArg] [Cha] S H K [Tle] [Oic] [Nle] [Aib][4-Cl-F] {COOH} 414 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00339 0.003785 Q R P[NMeArg] [Cha] S H K G [Aib] [Nle] P [4-Cl-F] {COOH} 418 {TDA} [AC4Abu][Aeea] [Aeea] 0.00014673 0.00025275 Q R P [NMeArg] [Cha] S H K G[aMePro] [Nle] P [4-Cl-F] {COOH} 498 {TDA} [AC4Abu] [Aeea] [Aeea]0.0000299 0.0001375 q R P [NMeArg] [Cha] S H K G [aMePro] [Nle] [Aib][4-Cl-F] {COOH} 545 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0013005 0.0036975 q RP [NMeArg] [Cha] S H K G [aMePro] [Nle] [BhPro] [4-Cl-F] {COOH} 551{TDA} [AC4Abu] [Aeea] [Aeea] 0.0002345 0.0011455 q R P [NMeArg] [Cha] SH K G [aMePro] [Nle] [4-Cl-F] {COOH} 417 {TDA} [AC4Abu] [Aeea] [Aeea]0.00599 0.008095 Q R P [NMeArg] [Cha] S H K G [BhPro] [Nle] P [4-Cl-F]{COOH} 415 {TDA} [AC4Abu] [Aeea] [Aeea] 0.003915 0.00401 Q R P [NMeArg][Cha] S H K G [Deg] [Nle] P [4-Cl-F] {COOH} 377 {TDA} [AC4Abu] [Aeea][Aeea] 0.0003875 0.000447 q R P [NMeArg] [Cha] S H K G [Oic] [Nle] P[4-Cl-F] {COOH} 379 {TDA} [AC4Abu] [Aeea] [Aeea] 0.007525 0.008375 Q R P[NMeArg] [Cha] S H K G [Oic] [Nle] P [BhPhe] {COOH} 380 {TDA} [AC4Abu][Aeea] [Aeea] 0.016665 0.00477 Q R P [NMeArg] [Cha] S H K G [Oic] [Nle]P [D-BhPhe] {COOH} 381 {TDA} [AC4Abu] [Aeea] [Aeea] 0.006115 0.005425 QR P [NMeArg] [Cha] S H K G [Oic] [Nle] P [D-4ClF] {COOH} 382 {TDA}[AC4Abu] [Aeea] [Aeea] 0.0004685 0.000682 Q R P [NMeArg] [Cha] S H K G[Oic] [Nle] [Aib] [4-Cl-F] {COOH} 390 {TDA} [AC4Abu] [Aeea] [Aeea]0.0001103 0.00057 Q R P [NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F]{COOH} 409 {TDA} [AC4Abu] [Aeea] [Aeea] 0.001003 0.000511 Q R P [NMeArg][Cha] S H K G [Oic] [Nle] [4-Cl-F] {COOH} 410 {TDA} [AC4Abu] [Aeea][Aeea] 0.002465 0.00268 Q R P [NMeArg] [Cha] S H K G [Oic] [Nle][D-4ClF] {COOH} 411 {TDA} [AC4Abu] [Aeea] [Aeea] 0.002105 0.00294 Q R P[NMeArg] [Cha] S H K G [Oic] [Nle] [Deg] [4-Cl-F] {COOH} 412 {TDA}[AC4Abu] [Aeea] [Aeea] 0.000614 0.0004365 Q R P [NMeArg] [Cha] S H K G[Oic] [Nle] [aMePro] [4-Cl-F] {COOH} 413 {TDA} [AC4Abu] [Aeea] [Aeea]0.0015275 0.001538 Q R P [NMeArg] [Cha] S H K G [Oic] [Tle] P [4-Cl-F]{COOH} 445 {TDA} [AC4Abu] [Aeea] [Aeea] 0.004285 0.00471 Q R P [NMeArg][Cha] S H K G [Oic] [Nle] [Pip] [4-Cl-F] {COOH} 487 {TDA} [AC4Abu][Aeea] [Aeea] 0.0018275 0.00819 Q R P [NMeArg] [Cha] S H K G [Oic] [Nle][BhPro] [4-Cl-F] {COOH} 540 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00041050.0012265 q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl-F] {COOH}541 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00093825 0.0019788 q R P [NMeArg][Cha] S H K G [Oic] [Nle] [aMePro] [4-Cl-F] {COOH} 542 {TDA} [AC4Abu][Aeea] [Aeea] 0.00519 0.016075 q R P [NMeArg] [Cha] S H K G [Oic] [Nle][BhPro] [4-Cl-F] {COOH} 549 {TDA} [AC4Abu] [Aeea] [Aeea] 0.001195 0.004q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Deg] [4-Cl-F] {COOH} 416 {TDA}[AC4Abu] [Aeea] [Aeea] 0.0001915 0.000346 Q R P [NMeArg] [Cha] S H K G[Pip] [Nle] P [4-Cl-F] {COOH} 499 {TDA} [AC4Abu] [Aeea] [Aeea]0.00002365 0.00008525 q R P [NMeArg] [Cha] S H K G [Pip] [Nle] [Aib][4-Cl-F] {COOH} 544 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0024225 0.0056075 q RP [NMeArg] [Cha] S H K G [Pip] [Nle] [BhPro] [4-Cl-F] {COOH} 361 {TDA}[AC4Abu] [Aeea] [Aeea] 0.0003085 0.000085967 Q R P [NMeArg] [Cha] S H KG P [Nle] P [4-Cl-F] {COOH} 437 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00066350.0007245 Q R P [NMeArg] [Cha] S V K G [Oic] [Nle] P [4-Cl-F] {COOH} 436{TDA} [AC4Abu] [Aeea] [Aeea] 0.000793 0.00147 Q R P [NMeArg] [Cha] S Y KG [Oic] [Nle] P [4-Cl-F] {COOH} 531 {TDA} [AC4Abu] [Aeea] [Aeea]0.0008515 0.00566 q R P [NMeArg] [Cha] S Y K G [Oic] [Nle] [Aib][4-Cl-F] {COOH} 459 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00007275 0.00023 Q RP [NMeArg] [NMeLeu] S H K G P [Nle] P [4-Cl-F] {COOH} 439 {TDA} [AC4Abu][Aeea] [Aeea] 0.00215 0.003205 Q R P [NMeArg] [Tle] S H K G [Oic] [Nle]P [4-Cl-F] {COOH} 468 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00002925 0.000165 QR P [NMeArg] L [aMeS] H K G P [Nle] P [4-Cl-F] {COOH} 464 {TDA} [AC4Abu][Aeea] [Aeea] 0.00005425 0.000377 Q R P [NMeArg] L [bAla] H K G P [Nle]P [4-Cl-F] {COOH} 467 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0001287 0.0005205 QR P [NMeArg] L [NhSerG] H K G P [Nle] P [4-Cl-F] {COOH} 450 {TDA}[AC4Abu] [Aeea] [Aeea] 0.000132 0.000313 Q R P [NMeArg] L [Pra] H K G P[Nle] P [4-Cl-F] {COOH} 423 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0006450.001182 Q R P [NMeArg] L S [bAla] K G P [Nle] P [4-Cl-F] {COOH} 469{TDA} [AC4Abu] [Aeea] [Aeea] 0.0001775 0.001329 Q R P [NMeArg] L S[rHis] K G P [Nle] P [4-Cl-F] {COOH} 449 {TDA} [AC4Abu] [Aeea] [Aeea]0.000394 0.00047 Q R P [NMeArg] L S H [1-Nal] G P [Nle] P [4-Cl-F]{COOH} 424 {TDA} [AC4Abu] [Aeea] [Aeea] 0.004145 0.017205 Q R P [NMeArg]L S H [bAla] G P [Nle] P [4-Cl-F] {COOH} 466 {TDA} [AC4Abu] [Aeea][Aeea] 0.0000461 0.0001925 Q R P [NMeArg] L S H [Nle] G P [Nle] P[4-Cl-F] {COOH} 448 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0001335 0.0003055 Q RP [NMeArg] L S H H G P [Nle] P [4-Cl-F] {COOH} 352 {TDA} [AC4Abu] [Aeea][Aeea] 0.0001085 0.00022983 Q R P [NMeArg] L S H K G [Oic] [Nle] P[4-Cl-F] {COOH} 403 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00263 0.008095 Q R P[NMeArg] L S H K G P [Nle] [2Nal] {COOH} 402 {TDA} [AC4Abu] [Aeea][Aeea] 0.0001002 0.0002465 Q R P [NMeArg] L S H K G P [Nle] [4-Cl-F]{COOH} 401 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0000816 0.0003515 Q R P[NMeArg] L S H K G P [Nle] [aMePro] [4-Cl-F] {COOH} 425 {TDA} [AC4Abu][Aeea] [Aeea] 0.0000937 0.000157 Q R P [NMeArg] L S H K G P [Nle] [bAla][4-Cl-F] {COOH} 426 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0001062 0.0003825 Q RP [NMeArg] L S H K G P [Nle] [BhPro] [4-Cl-F] {COOH} 429 {TDA} [AC4Abu][Aeea] [Aeea] 0.00006635 0.00009355 Q R P [NMeArg] L S H K G P [Nle][Pip] [4-Cl-F] {COOH} 287 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00007490.00024185 Q R P [NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH} 470 {TDA}[AC4Abu] [Aeea] [Aeea] 0.00003795 0.0001495 Q R P [NMeArg] L S H K G P[Nle] P [2Nal] {COOH} 456 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00002920.000178 Q r P [NMehArg] [aMeLeu] S H K G P [Nle] P [4-Cl-F] {COOH} 384{TDA} [AC4Abu] [Aeea] [Aeea] 0.0009565 0.00435 Q r P [NMehArg] [BLeu] SH K G P [Nle] P [4-Cl-F] {COOH} 435 {TDA} [AC4Abu] [Aeea] [Aeea]0.0004605 0.0011465 Q r P [NMehArg] [Cha] S H K G P [Nle] P [4-Cl-F]{COOH} 471 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0000229 0.0000974 Q R P[NMehArg] L S H K G P [Nle] P [2Nal] {COOH} 526 {TDA} [AC4Abu] [Aeea][Aeea] 0.002025 0.0072 q R P E [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl-F]{COOH} 527 {TDA} [AC4Abu] [Aeea] [Aeea] 0.1075 0.227 q R P e [Cha] S H KG [Oic] [Nle] [Aib] [4-Cl-F] {COOH} 477 {TDA} [AC4Abu] [Aeea] [Aeea]0.000423 0.000691 Q R P E L S H K G P [Nle] P [4-Cl-F] {COOH} 427 {TDA}[AC4Abu] [Aeea] [Aeea] 0.0001705 0.000313 Q R P P L S H K G P [Nle] P[4-Cl-F] {COOH} 465 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0000692 0.0001745 Q RP Q L S H K G P [Nle] P [4-Cl-F] {COOH} 430 {TDA} [AC4Abu] [Aeea] [Aeea]0.0012155 0.002835 Q r P r [Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH}480 {TDA} [AC4Abu] [Aeea] [Aeea] r 0.00009985 0.000271 Q [hArg] P [hArg][Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 481 {TDA} [AC4Abu] [Aeea][Aeea] r 0.0001765 0.000341 q [hArg] P [hArg] [Cha] S H K G [Oic] [Nle]P [4-Cl-F] {COOH} 479 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0004085 0.0008055 rQ [hArg] P [NMeArg] [Cha] S H K G [Oic] [Nle] P [4-Cl-F] {COOH} 348{TDA} [AC4Abu] [Aeea] [Aeea] 0.004915 0.007455 R Q [Nle] P [hArg] [Cha]A A K G [Oic] [Nle] P [4-Cl-F] {COOH} 343 {TDA} [AC4Abu] [Aeea] [Aeea]0.001395 0.00124 R Q [Nle] P [hArg] [Cha] S A K G [Oic] [Nle] P [4-Cl-F]{COOH} 564 {TDA} [AC4Abu] [Aeea] [Aeea] 0.000331 0.0014925 R Q [Nle] P[hArg] [Cha] S A K G [Pip] [Nle] P [4-Cl-F] {COOH} 339 {TDA} [AC4Abu][Aeea] [Aeea] 0.00255 0.004525 R Q [Nle] P [hArg] [Cha] S H K G [Oic][Nle] P [4-Cl-F] {COOH} 351 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0015950.001805 R Q R P [hArg] [Cha] A A [Nle] G [Oic] [Nle] P [4-Cl-F] {COOH}340 {TDA} [AC4Abu] [Aeea] [Aeea] 0.0003445 0.000676 R Q R P [hArg] [Cha]S A [Nle] G [Oic] [Nle] P [4-Cl-F] {COOH} 336 {TDA} [AC4Abu] [Aeea][Aeea] 0.000714 0.0009865 R Q R P [hArg] [Cha] S H [Nle] G [Oic] [Nle] P[4-Cl-F] {COOH} 565 {TDA} [AC4Abu] [Aeea] [Aeea] r 0.001023 0.00282 Q RP [hArg] [NMeLeu] S H K G [Pip] [pI-Phe] P [D-Bip] {COOH} 345 {TDA}[AC4Abu] [Aeea] [Aeea] 0.00451 0.005785 R Q R P [Nle] [Cha] A A K G[Oic] [Nle] P [4-Cl-F] {COOH} 344 {TDA} [AC4Abu] [Aeea] [Aeea] 0.003460.005575 R Q R P [Nle] [Cha] A H K G [Oic] [Nle] P [4-Cl-F] {COOH} 350{TDA} [AC4Abu] [Aeea] [Aeea] 0.0002855 0.0006235 R Q R P [Nle] [Cha] S AK G [Oic] [Nle] P [4-Cl-F] {COOH} 335 {TDA} [AC4Abu] [Aeea] [Aeea]0.000315 0.000964 R Q R P [Nle] [Cha] S H K G [Oic] [Nle] P [4-Cl-F]{COOH} 346 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00277 0.002455 R Q R P K [Cha]A A [Nle] G [Oic] [Nle] P [4-Cl-F] {COOH} 341 {TDA} [AC4Abu] [Aeea][Aeea] 0.000561 0.001265 R Q R P K [Cha] S A [Nle] G [Oic] [Nle] P[4-Cl-F] {COOH} 337 {TDA} [AC4Abu] [Aeea] [Aeea] 0.00208 0.00212 R Q R PK [Cha] S H [Nle] G [Oic] [Nle] P [4-Cl-F] {COOH} 539 {TDA} [AC4Abu][NPeg11] q R P 0.00073525 0.003545 [NMeArg] [Cha] S H K G [Oic] [Nle][Aib] [4-Cl-F] {COOH} 532 {TDA} [AC4Abu] q R P 0.00068225 0.00299[NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl-F] {COOH} 507 {TDA}[Aeea] [Aeea] Q R P 0.00007945 0.000245 [NMeArg] L S H K G P [Nle] P[4-Cl-F] {COOH} 538 {TDA} d [Aeea] [Aeea] q R P 0.00052525 0.001891[NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl-F] {COOH} 546 {TDA} D[Aeea] [Aeea] q R P 0.000478 0.0012335 [NMeArg] [Cha] S H K G [Oic][Nle] [Aib] [4-Cl-F] {COOH} 537 {TDA} E [Aeea] [Aeea] q R P 0.000280220.0010833 [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib] [4-Cl-F] {COOH} 547{TDA} e [Aeea] [Aeea] q R P 0.00040425 0.0013333 [NMeArg] [Cha] S H K G[Oic] [Nle] [Aib] [4-Cl-F] {COOH} 511 {TetDDA} [AC4Abu] [Aeea] 0.0001810.0009685 [Aeea] Q R P [NMeArg] L S H K G P [Nle] P [4-Cl-F] {COOH}

Example 11. Immunoglobulin Fc-Apelin Peptide Conjugates

Four modified apelin polypeptides of the invention (SEQ ID NOs: 8-11)were used for site selective conjugation to an engineered Fc region of ahuman IgG. The peptides were conjugated to the Fc region through abromoacetyl-NPeg11 linker attached at the amino terminus of thepeptides. By reacting the bromoacetamide portion of the linker with afree cysteine residue in the Fc protein, a covalent thioether linkagewas formed as illustrated below.

The sequences of the four peptides are shown in Table 17. The human IgGFc protein sequence is shown below.

Human IgG Fc Sequence (SEQ ID NO: 716) 1 MDKTHTCPPCPAPELLGGPSVFLFPPKPKD30 31 TLMISRTPEVTCVVVDVSHEDPCVKFNWYV 60 61DGVEVHNAKTKPREEQYNSTYRVVSVLTVL 90 91 HQDWLNGKEYKCKVSNKALPAPIEKTISKA 120121 KGQPREPQVYTLPPSRDELTKNQVSLTCLV 150 151KGFYPSDIAVEWESNGQPENNYKTTPPVLD 180 181 SDGSFELYSKLTVDKSRWQQGNVESCSVMH210 211 EALHNHYTQKSLSLSPGKThe underlined C indicates the site of an engineered cysteine and thepoint of covalent attachment of the modified apelin polypeptide. In thehomodimer C7-C7 and C10-C10 are intermolecular disulfides, whereasC42-C102 and C148-C206 are intramolecular disulfides.

TABLE 17 Amino Acid Sequences of Modified Apelin Polypeptides forSite-Selective Fc Conjugation SEQ ID NO: AMINO ACID SEQUENCE 8QrP[hArg][aMeLeu]SHKGP[Nle]P[4-Cl-F]{COOH} 9 QR[aMePro][hArg][aMeLeu]SHKGP[Nle]P[4-Cl-F]{COOH} 10 Q[NMehArg]P[NMeArg][aMeLeu]SHKGP[Nle]P[4-Cl-F]{COOH} 11 Q R P [NMeArg] [Cha] S H K G [Oic] [Nle] [Aib][4-Cl-F]{COOH}

The Fc protein forms a homodimer upon folding and thus contains twoengineered cysteine reactive sites per folded protein. Each Fc-homodimerconjugate will ideally contain two copies of the modified apelinpolypeptide. Each of the conjugates were evaluated for efficacy inactivating the rat and human APJ receptors as measured by the GTPγSassay (see Example 3 for methods). The conjugates were also tested forinduction of histamine release from rat mast cells (see Example 7 formethods). The results of these experiments are summarized in Table 18below. The data for the unconjugated peptides are also shown forcomparison. As can be seen from the results in the table, conjugation ofthe apelin peptides did not significantly affect the peptide's potencyin activating the APJ receptor. In addition, none of the Fc-peptideconjugates induced histamine release from rat mast cells. In one case,the Fc-conjugate eliminated the histamine release activity of theunconjugated peptide (see Fc-peptide conjugate #4).

TABLE 18 Comparison of Fc-Apelin Peptide Conjugates to UnconjugatedApelin Peptides EC50 Human EC50 SEQ APJ Rat APJ ID SEQUENCE/ ReceptorReceptor Histamine Compound NO: DESCRIPTION (μM) (μM) release?Acetylated 70 {Acetyl-NH} 0.0001925 0.009735  No version of Q[r]P[hArg]peptide in [aMeLeu]SHK Fc-peptide GP[Nle] conjugate P[4-Cl-F] #1 {COOH}Fc-peptide — human IgG Fc 0.000258  0.00107   No conjugate conjugate #1of apelin peptide of SEQ ID NO: 8 Acetylated 69 {Acetyl-NH}QR 0.00004170.0003135 No version of [aMePro][hArg] peptide in [aMeLeu] Fc-peptideSHKGP conjugate [Nle]P[4-Cl-F] #2 {COOH} Fc-peptide — human IgG Fc0.000208  0.000304  No conjugate conjugate #2 of apelin peptide of SEQID NO: 9 Acetylated 95 {Acetyl-NH} 0.000177  0.00393   No version ofQ[NMehArg] peptide in P[NMeArg] Fc-peptide [aMeLeu] conjugate SHKGP #3[Nle]P[4-Cl-F] {COOH Fc-peptide — human IgG Fc 0.000319  0.000682  Noconjugate conjugate #3 of apelin peptide of SEQ ID NO: 10 Acetylated 92{Acetyl-NH} 0.000679  0.0032275 Yes version of QRP[NMeArg] peptide in[Cha]SHK Fc-peptide G[Oic][Nle] conjugate [Aib][4-Cl-F] #4 {COOH}Fc-peptide — human IgG Fc 0.000746  0.00137   No conjugate conjugate #4of apelin peptide of SEQ ID NO: 11

Throughout this specification various publications, patents and patentapplications have been referenced. The disclosures of these documents intheir entireties are hereby incorporated by reference into thisapplication. The reference to such documents, however, should not beconstrued as an acknowledgment that such documents are prior art to theapplication.

What is claimed:
 1. A method for treating heart failure in a subject inneed thereof comprising administering to the subject an isolatedpolypeptide comprising the amino acid sequence SEQ ID NO: 16; andwherein the polypeptide is conjugated to polyethelyene glycol by3-mercaptopropanoic acid.
 2. The method of claim 1, wherein dP/dt_(max),and/or ejection fraction is increased in the subject followingadministration of the polypeptide.
 3. The method of claim 1, whereinsystolic or diastolic function is improved in the subject followingadministration of the polypeptide.
 4. The method of claim 1, wherein theheart failure is heart failure with reduced ejection fraction.
 5. Amethod of improving cardiac contractility in a subject having acardiovascular condition comprising administering to the subject thepolypeptide of claim 1, wherein the polypeptide comprises the amino acidsequence SEQ ID NO: 16; and wherein the polypeptide is conjugated topolyethylene glycol by 3-mercaptopropanoic acid; and wherein cardiaccontractility is improved in the subject following administration.
 6. Amethod of increasing ejection fraction in a subject having acardiovascular condition comprising administering to the subject thepolypeptide of claim 1, wherein the polypeptide SEQ ID NO: 16, andwherein the polypeptide is conjugated to polyethelyene glycol by3-mercaptopropanoic acid and wherein the ejection fraction is increasedfollowing administration of the polypeptide.